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M.Sc. (Final) Chemistry

PAPER –II : BIOINORGANIC, BIO ORGANIC & BIOPHYSICAL

CHEMISTRY

BLOCK – I

Unit -1 : Biological Cell

Unit-2 : Metal Ions in Biological systems

Unit-3 : Bioenergetics

Author – Dr. Purushottam B. Chakrawarti

Dr. Aruna Chakrawarti

Editor - Dr. Anuradha Mishra

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SUMMARY

Cell is the structural and functional unit of life. Eukaryotic cells

contain many membrane bounded organelles that carryout specific cellular

processes. Chief organelles are cell membrane, nucleus, mitochondria,

endoplasmic reticulum, lysosomes and centrioles. Important biomolecules

are proteins, enzyme, DNA and RNA. An essential role of biomolecules is to

allow movement of all compounds necessary for the normal function of a

cell across the membrane barrier.

Metal compounds are closely related to the life process. Among these

compounds are haemoglovin, chlorophyll, numerous haematin enzymes,

metal activated enzymes, vitamin B12 and those vital but poorly understaood

complexes which play an important role in the metabolism of the metal ions.

Na+/K

+ pump is Na

+/K

+ ATPASE and is found to maintain both magnitude

and direction of transmembrane concentration gradients of these ions.

Haemoglobin, myoglovin and haemerythrin play important part in the

transportation of oxygen.

Bioenergetics or biochemical thermodynamics is the study of energy

changes in biochemical reactions. Free energy, G, is the useful energy also

known as the chemical potential. In the living cells, the principal high

energy intermediate or carrier compound is ATP.

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UNIT – 1 BIOLOGICAL CELL

Structure

1.1 Introduction

1.2 Objectives

1.3 Biological Cell and its Structure

1.4.1 Proteins

1.4.2 Enzymes

1.4.3 DNA and RNA in Living Systems

1.4.4 Helix Coil Transition

1.5 Cell Membrane and Transport of Ions

1.5.1 Structure and Function of Cell-Membrane

1.5.2 Ion Transport Through Cell- Membrane Transport

1.5.3 Irreversible Thermodynamic Treatment of Membrane Transport

1.5.4 Nerve Conduction

1.6 Let Us Sum Up

1.7 Check Your Progress : The Key

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1.1 INTRODUCTION

Cell is the structural and functional unit of life. In multicellular

organisms like man, animals and trees special functions are performed by

special structures called organs, which are themselves composed of tissues.

Tissues are groups of cells, which perform same functions and are also

similar in structure. (The structure of a typical animal cell is shown in Fig.

1.1). Nucleus of the cell controls its activities. Messages are passed on from

the nucleus through the pores in the nuclear membrane into cytoplasm for

coordination of various cellular activities, including the synthesis of proteins

and enzymes required for life functions. The chromosomes, long and thin

threads forming the complex fibrous 'chromatin network', 'resting nucleus',

are the carrier of the genetic information encoded in the genes for different

traits, e.g., eye colour, height, facial characters and blood group. They also

determine an individual's sensitivity to the herpes virus and the polio virus,

susceptibility to cancer, and to a host of other human diseases. Genes that

prevent cancer (suppressor genes) and those involved in cancer (oncogenes)

are also located on the chromosomes.

Each gene consists of a very long molecule called deoxyribonucleic

acid (DNA). DNA is made up of many small subunits called nucleotides.

There are four kinds of nucleotides known, each containig a different

nitrogeneous base in the unit, represented by four letters A (adenine), G.

(guanine), T. (thymine) and C (cystosine). These four letters, A, G, T and C

constitute the genetic dictionary, and their combinations and sequences

encode all the genetic information in all forms of life. It is the sequence of

subunits in a DNA molecule which is characteristic of a particular gene, i.e.,

the differences between genes lie in the precise sequence of nucleotides,

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which confers on each gene the unique property to specify its genetic code.

Gene expression is controlled by controlling regions of DNA, the so called

regulatory genes. Genes carry instructions for the synthesis of proteins,

using 20 different amino acids. Genetic code is a triplet out of the four

letters A, G, T, C [total (4)3 = 64 triplets possible]. Gene give instructions to

amino acids to line up in a certain sequence through m-RNA (massager -

Ribonucleic acid); which along with r-RNA (ribosomal-RNA) and t-

RNA (transfer-RNA) synthesise protein in cytoplasm.

Structure and function of biological cell are discussed in this unit.

Fig. 1.1 : Structure of an animal cell. In the centre of the cell is a

circular body called the nucleus, which controls the cell's activities.

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1.2 OBJECTIVES

The main objectives of this unit is to discuss biological cell, its

structure and function. After going through this unit you would be able to :

describe biological cell and its structure,

discuss cell membrane and transport of ions though it,

understand irreversible thermodynamic treatment of membrane

transport, and

underline nerve conduction.

1.3 BIOLOGICAL CELL AND ITS STRUCTURE

All organisms are built from cells. All animal tissues including human

are also organized from collections of cells. Thus cell is the fundamental unit

of life. If cell dies, tissue dies and it cannot function. Modern cell theory can

be underlined (into the following fundamental statement:

Cells make up all living matter and the genetic information required

during the maintenance of existing cells and the production of new

cells passes from one generation to the other next generation. The

chemical reactions of an organism that is its metabolism, both

anabolism and catabolism, takes place in the cells.

In general two type of cells exist in nature :

(a) The Prokaryotic cell and (b) the Eukaryotic cell.

(a) The Prokaryotic cell

Typical prokaryotic cells (Greek : Pro=before and Karyon-nucleus)

include the bacteria and cyano Karyon-nucleus) include the bacteria and

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cyano bacteria. The most studied prokaryotic cell is Escherichia Coli (E.

Coli), which–

Has a minimum of internal organization and is smaller in size. It does

not have any membrane bound organelles, its genetic material is not

enclosed by a nuclear membrane and DNA is not complexed with histones.

Histones are not found in prokaryotic cells.

Its respiratory system is closely associated with its plasma membrane

and sexual reproduction does not involve mitosis on meiosis.

(b) Eukaryotic Cells

The eukaryotic cells (Greek : Eu=true and karyon=nucleus) include

the protests, fungi, plants and animals including humans. Cells are larger in

size (Refer Fig.1.1) and have considerable degree of internal structure with a

large number of distinctive membrane enclosed having specific functions.

Nucleus is the site for informational components collectively called

chromatin and its sexual reproduction involves both mitosis and meiosis.

The respiratory site is the mitochondria. In the plant cells, the site of the

conversion of radiant energy to chemical energy is the highly structural

chloroplasts.

Essential differences of prokaryotic and Eukaryotic cells are given in

Table. 1.1

Table 1.1 Differences between prokaryotic and Eukaryotic cells

Prokaryotic Cell Eukaryotic Cell

1. Smaller in size 1 to 10 m 1. Larger in size 10 to 100 m or more

2. Mainly unicellular 2. Mainly multicelllar (with few exceptions).

Several different types present.

3. Single membrane, surrounded

by rigid cell wall

3. Lipid bilayer membrane with proteins

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4. Anaerobic or aerobic 4. Aerobic

5. Not well defined nucleus, only a

nuclear zone with DNA

Histones absent

5. Nucleus well defined, 4 to 6 m in

diameter, contains DNA and surrounded

by a perinuclear membrane Histones

present.

6. No nuclei 6. Nucleolus present, rich in RNA

7. Cytoplasm contains no cell

organelles

7. Membrane bound cell organelles are

present.

8. Ribosomes present free in

cytoplasm

8. Ribosomes studded on outer surface of

endoplasmic reticlum present.

9. Mitochondria absent. Enzymes

of energy metabolism bound to

membrane

9. Mitochondria present "Power house" of

the cell. Enzymes of energy metabolism

are located in mitochondria.

10 Golgi apparatus absent. Storage

granules with polysaccharides.

1

0

Golgi apparatus present-flattened single

membrane vesicles.

11. Lysosomes-absent 1

1.

Lysosomes present-single membrane

vesicle containing packets of hydrolytic

enzymes.

12 Cell division usually by fission,

no mitosis

1

2

Cell division-by mitosis

13 Cytoskeleton-absent 1

3.

Cytoskeleton-present

14 RNA and protein Synthesis in

same compartment

1

4.

RNA synthesized and processed in

nucleus. Proteins synthesized in

cytoplasm.

15. Examples are bacteria,

cyanobacteria, rickettsii

1

5.

Examples : Protists, fungi, plants and

animal cells.

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Structure of a cell

Eukaryotic cells contain many membrane-bounded organelles that

carryout specific cellular processes. Chief organelles and their functions are

as follows :

1. Cell Membrane

The boundary of every cell is limited by a membrane (10 mm thick)

known as cell, unit or plasma membrane. The membrane is believed to be

formed by the ingredients of the plasma (cytoplasm).

Electron micrographs of membranes of various origin are remarkably

similar and show that the membrane consists of two dark bands each of 20Aº

thickness separated from a light band of about 35Aº as its thickness. Thus

the average overall thickness of the membrae is 75Aº although the value

varies from 70Aº to 100Aº.

Fig 1.2 : A cross section of cell membrane

The dark bands are composed of proteins, the nature of which are still

obscure, although some may be enzymatic in nature. On the other hand, the

light band is composed of phospholipids and cholesterol. The phospholipid

molecules in the light band are arranged in two rows in a way that the

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phosphate containing ends, known as polar or hydrophilic ends point to the

outside while the non-polar or hydrophobic chains point to the inside.

The surface of the cell-membrane bears pores, of internal diameter

3Aº and length about 75Aº, which lead from cytoplasm to the exterior.

2. Nucleus

Nucleus is the heaviest particulate component of the cell. It lies

approximately in the middle of the cell. Except mature mammalian

erythrocytes, nucleus is found in almost all cells. Its membrane, having some

pores, consists of two layers (outer and inner) separated from each other by

the so-called perinuclear space or perinuclear cisterns (150Aº). The pores on

nuclear membrane are of higher diameter than that of the cell membrane and

allows very big molecules of nucleic acids and proteins to pass through. The

nuclear pores interlink the nuclear material with the cytoplasm.

The nucleus contains more than 95% of the cell's DNA and is the

control cenre of the eukaryotic cell. Its important components are :

(i) Nuclear envelope : A double membrane structure called the nuclear

envelope separates the nucleus from the cytosol.

(ii) Nuclear pore complexes : are embedded in the nuclear envelope.

These complex structures control the movement of proteins and the

nucleic acid ribonucleic acids (RNAs) across the nuclear envelope.

(iii) Chromatin : DNA in the nucleus is coiled into a dense mass a

irregular clumps of thread like work, called chromatin, so named

because it is stained darkly with certain dyes.

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(iv) Chromosomes – Immediately before the cell division, the chromatin

organizes into simple small thread like structures known as

chromosomes which will eventually be distributed equally to each

daughter cell. After the cell division, the chromosomes of each

individual daughter cell uncoil and disperse in their respective nuclei

into chromatin net-work. Chromosomes are composed of

nucleoprotein molecules, the DNA molecules act as prosthetic group.

As mentioned earlier most of the cellular DNA is present in the

nucleus and further almost exclusively in chromosomes.

(v) Nucleolus : A second dense mass closely associated with the inner

nuclear envelope is called nucleolus.

(vi) Nucleoplasm : Nucleoplasm of nucleus contain various enzymes such

as DNA polymerases, and RNA polymerases, for m-RNA and t-RNA

synthesis.

Functions :

(i) DNA Replication and RNA transcription of DNA occur in the

nucleus. Transcription is the first step in the expression of genetic

information and is the major metabolic activity of the nucleus.

(ii) The nucleolus is non-membranous and contains RNA polymerase,

RNA ase, ATP ase and other enzymes, but not DNA polymarase.

Nucleolus is the site of synthesis of ribosomal RNA (r-RNA)

(iii) Nucleolus is also the major site where ribosome subunits are

assembled.

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3. Mitochondria

These are the largest particulate components of the cytoplasm, and

represent upto 15-20% of the dry weight of the liver cell. The contribute

about 35 per cent of the total protein of the liver. In addition to proteins they

contain lipids (25%), most of which (about two-third) are phosopholipids,

and a relatively small amount of nucleic acids.

Fig 1.3 : (A) Mitochondrion : Shows half split (B) Cross section of a

to show the inner membrane with cristae mitochondria

The number of mitochondria in a cell varies dramatically. Some algae

contain only one mitochondrion, whereas the protozoan Chaos contain half a

million. A mammalian liver cell contains from 800 to 2500 mitochondria.

They vary greatly in size. A typical mammalian mitochondrion has a

diameter of 0.2 to 0.8 and a length of 0.5 to 1.0 m. The shape of

mitochondrion is not static. Mitochondria assume many different shapes

under different metabolic conditions.

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Structure and Functions

The mitochondrion is bounded by two concentric membranes that

have markedly properties and biological functions.

Mitochondrial Membranes:

(a) Outer mitochondrial membrane : The outer mitochondrial

membrane consists mostly of phospholipids and contains a considerable

amount of cholesterol. The outer membrane also contains many copies of the

protein called "Porin".

Functions of Porin and other Proteins:

(i) These proteins form channels that permit substances with

molecular weights of less than <10,000 to diffuse freely across the

outer mitochondrial membrane.

(ii) Other proteins in the outer membrane carry out various reactions in

fatty acid and phospholipid biosynthesis and are responsible for

some oxidation reactions.

(b) Inner mitochondrial membrane : The inner mitochondrial

membrane is very rich in proteins and the ratio of lipid to proteins is only

0.27:1 by weight. It contains high proportion of the phospholipid cardiolipin.

In contrast to outer membrane, the inner membrane is virtually impermeable

to polar and ionic substances. These substances enter the mitochondrion only

through the mediation of specific transport proteins.

Cristae: The inner mitochondrial membrane is highly folded. The

tightly packed inward folds are called "cristae". It is now known that

mitochondria undergo dramatic changes when they switch over from

resting state to a respiring state. In the respiring state, the inner

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membrane is not folded into cristae, rather it seems to shrink leaving

much more voluminous intermembrane space.

(c) Inter membrane space: The space between the outer and inner

membranes is known as the "intermembrane space". Since the outer

membrane is freely permeable to small molecules, the intermembrane space

has about the same ionic composition as the cytosol.

(d) Mitochondrial matrix: The region enclosed by the inner membrane

is known as the mitochondrial matrix.

4. Endoplasmic Reticulum

In many cells a system of Internal membranes termed the endoplasmic

reticulum is found within the cytoplasm. Such membranes are generally

constructed of protein lipid double layers and are very well developed in

tissues with active protein synthesis, viz. in exocrine cell of the pancreas.

The Golgi bodies may serve as a means of producing and maintaining this

internal membrane. The interior of the enploplasmic reticulum appears to be

connected with perinuclear space, and sometimes also with the exracellular

space (through pores in the cell surface). The cisternae (enclosed spaces) of

the endoplasmic reticulum play a role in the exchange of material between

the cell and the extracellular fluid. The exchange of material takes place by

the processes of diffusion, active transport and phatocytosis (pinocytosis). In

the latter process, a small area of the cellar membrane engulfs the material,

forming a vacuole, which is then transported to the interior of the cell. The

cellular membrane re-forms, the entering vacuole disintegrates within the

cell and thus mixes its contents with the cytoplasm. On the other hand, small

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portions of the membrane of the endoplasmic reticulum can leave cell and

spill their content to the exterior.

These organelles are involved in protein synthesis, transport,

modification, storage and secretion.

Varying in shape, size and amount, the endoplasmic reticulum (ER)

extends from the cell membrane, coarts the nucleus, surrounds the

mitochondria and appears to connect directly to the golgi apparatus. These

membranes and the aqueous channels they enclose, called "cisernae", are

thus important organelles.

There are two kinds of endoplasmic reticulum (ER)

(i) Rough surfaced ER, also known as "argastoplasm". They are

coated with ribosomes. Near the nucleus, this type of ER merges

with the outer membrane of the nuclear envelope.

(ii) Smooth surfaced ER : They do not have attached ribosomes.

Golgi complexes (or Golgi apparatus) are also called "Dictyosomes".

Each eukaryotic cell contains a unique stack of smooth surfaced

compartments or cisternae that make-up the Golgi complex. The ER is

usually closely associated with the Golgi complexes, which contain

flattened, fluid filled Golgi sacs.

The Golgi complex has a "Proximal or 'Cis' compartment, a "medial"

compartment and a 'distal' or 'trans' compartment.

Recent evidence suggests strongly that the complex serves as a unique

sorting device that receives newly synthesized proteins, all containing signal

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or transit peptides from the ER. It is interesting to note that those proteins

with no signal or transit peptides regions are rejected by the Golgi apparatus

without processing it further and remain as cytoplasmic protein.

Functions :

(i) On the proximal or 'cis' side, the golgi complexes receive the

newly synthesized proteins by ER via transfer vesicles.

(ii) The posttranslational modifications take place in the golgi lumen

(median part) where the carbohydrates and lipid precursors are

added to proteins to form glycoproteins and lipoproteins

respectively.

(iii) On the distal or 'trans' side they release proteins via modified

membranes called "secretory vesicles". these secretory vesicles

move to and fuse with the plasma membrane where the contents

may be expelled by a process called "exocytosis".

5. Lysosomes

Lysosomes are particles intermediate in size (mean diameter =0.4 )

between microsomes and mitochondria. These are surrounded by a

lipoprotein membrane. Because of their richness in many hydrolytic

enzymes, they were named lysosomes. The various hydrolytic enzymes

(digestive enzymes) of the lysosomes break down fats, proteins, nucleic

acids and other large molecules into smaller molecules which are capable of

being metabolised by the mitochondrial enzymes. The various hydrolytic

enzymes present in lysosomes include the acid phosphatase, cathepsin ( a

protease), collagenase, ribonuclease, deoxyribonuclease, -glucuronidase,

-galactosidase, -mannosidase, -glucosidase, -N-acetyl glucosa-

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minidase, phosphoprotein phosphatase, aryl sulphatase, and others.

Hydrolases in lysosomes are separated from their substrates by means of

lysosomal membrane and only when the latter is ruptured the various

hydrolases enter the cytoplasm and exercise their specific functions. This

occur only under conditions of cytolysis which include fat

Fig. 1.4 – The lysosome

solvents, detergents, proteases, lecithinase, inadequate osmotic protection,

freezing and thawing, sanic vibrations, acid pH and high temperature.

The hydrolases of lysosomes completely destroy the foreign material

like bacteria whe the latter enter the cell and thus lysosomes also provide a

sort of protection to the cells and thus to the body.

6. Centrioles

Two short cylindrical structures, known as centrioles, are found to lie

one side of the nucleus. These are present at right angles to each other and

are not bound by any membrane. These help in the equal division of the

chromosomes by pulling them apart and thus are responsible for equally

distributing the characters in the offsprings.

1.4 FUNCTIONS OF BIOMOLECULES

Proteins are highly complex, natural compounds, composed of a large

number of different aminoacids i.e. they are naturally occurring

polypetides. Proteins are the chief constituents of protoplasm in all the living

INTACT LYSOSOME

ENZYMES BOUND AND

INACCESSIBLE

INJURED LYSOSOME AND HENCE ENZYMES

ACCESSIBLE

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cells; but on the whole, animals have relatively much more proteins than

plants in which cellulose predominaes. Among animals the mammales are

constituted largely of proteins (eg. skin, hair, nails, haemoglobin, muscles

etc). Antibodies, enzymes, some hormones, viz. insulin are proteinous in

nature. It is very important to note that the tissue proteins of any two of the

individuals are not identical, excep for two twins. Due to this characteristic,

proteins help in protecting the body by the attack of foreign toxic proteins

and virus; the latter are partially proteins.

The building blocks of proteins are the amino acids. An amino acid is

an organic acid in which the carbon next to the –COOH group (called an

alpha carbon) is also bound to an – NH2 group. In addition, the alpha carbon

is bound to a side-chain (R), which is different in each amino acid.

H

H2N C COOH

R

(side-chain)

The aminoacids differ from one another only in the side chain; for

example, the R in alanine has one carbon, while in leucine it has four

carbons. Table 1.2 shows that the properties of the various amino acids

depend on the chemical composition of their side chains; for example, lysine

and arginine are basic because their side chains contain an extra amino

group, and the acidic amino acids (glutamic and aspartic acids) contain an

extra carboxyl group.

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TABLE 1.2 THE TWENTY AMINO ACIDS

Type of Amino Acid 3-letter Symbol 1-Letter Symbol

Hydrophobic

(Aliphatic Side Chain)

Glycine Gly G

Alanine Ala A

Valine Val V

Leucin Leu l

Isoleucine lle l

Basic (Diamino)

Arginine Arg R

Lysine Lys K

Acidic (Dicarboxylic)

Glutamic acid Glu E

Aspartic acid Asp D

Amide-Containing

Glutamine Gln Q

Asparagine Asp N

Hydroxyl

Threonine Thr T

Serine Ser. S

Sulfur-Containing

Cysteine Cys C

Methionine Met. M

Aromaic

Phenylalanine Phe F

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Type of Amino Acid 3-letter Symbol 1-Letter Symbol

Tyrosine

Heterocyclic

Tryptophan Trp W

Proline Pro P

Histidine His H

The biological importance of proteins can be judged by the fact that

the animals can live for a long time without fat or carbohydrate, but not

without protein. Proteins mainly supply new tissues, repair working parts

and make up the loss (e.g. as gland secretions) in the vital processes. Only

the plants can built up proteins from inorganic materials, like nitrates,

ammonium sulphate, carbondioxide and water, while most of the animals

derive them mainly from plants and some other animals.

(a) Some proteins act as hormones and hence regulate various metabolic

processes, e.g. insulin is responsible for maintaining blood sugar

levels.

(b) Some proteins function as biological strctural materials, viz. collagen

in connective tissue and kera tin in hair,

(c) Some proteins (enzymes) function as catalysts for biological

reactions.

(d) Haemoglobin (a protein) acts as oxygen carrier in mammals.

Haemocyanin function similarly for shell-fish.

(e) Some blood proteins function to form antibodies which provide

resistance to diseases.

(f) Nucleoproteins form the important constituents of the genes that

supply and transmit genetic massages in cell divisions.

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1.4.2 Enzymes

The cell may be compared to a minute laboratory capable of carrying

out the synthesis and breakdown of numerous substances. These processes

are carried out by enzymes at normal body temperature, low ionic strength,

low pressure, and a narrow range of pH.

Enzymes are biological catalysts. A catalyst is a substance that

accelerates chemical reactions but that is not itself modified in the process,

so that it can be used again and again. The vast majority of enzymes are

proteins. In fact, until 1985 it was believed that all enzymes were proteins.

We now know that some RNA molecules may also have catalytic activities

and must thus be considered enzymes too (see below).

Enzymes are the largest and most specialized class of protein

molecules. More than a thousand different enzymes have been identified;

many of them have been obtained in pure, and even crystalline, condition.

Enzymes represent one of the most important products of the genes

contained in the DNA molecule. The complex network of chemical reactions

which are involved in cell metabolism is directed by enzymes.

Enzymes (E) are proteins with one or more loci, called active sites, to

which the substrate (S) (i.e., the substance upon which the enzyme acts)

attaches. The substrate is chemically modified and converted into one or

more products (P). Since this is generally a reversible reaction, it may be

expressed as follows :

E+S = [ES] = E+P

where [ES] is an inermediary enzyme-sbsrate complex. Enzymes accelerate

the reaction until an equilibrium is reached. They are so efficient that the

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reaction may proceed from 108 to 10

u times faster than in the noncatalyzed

condition.

A very important feature of enzyme activity is that it is substrate-

specific; i.e., a particular enzyme will act only on a certain substrate. Some

enzymes have nearly absolute specificity for a given substrate and will not

act on even very closely related molecules.

Some enzymes require the presence of a co-factor for their activity.

This may be a metal or a prosthetic group, in the case of conjugated proteins.

Some enzymes reqire the presence of small molecules called coenzymes. For

example, dehydrogenases require the presence of small molecules called

coenzymes. For example, dehydrogenases require a nicotinamide-adenine

dinucleotide (NAD+) or an NADP

+ molecule (with an additional phosphate)

to function. The reaction is as follows :

Substrate+NAD+Enzymeoxidized substrate+NADH and

H++Enzyme. The two electrons gained by NADH can then be transferred to

a second molecule, which will become reduced (i.e. it gains electrons).

In the cell the energy-producing enzymes use NAD as coenzyme, the

synthetic processes, however, use NADPH as a hydrogen donor. In many

coenzymes, as in NAD+ (which contain nicotinamide), the essential

components are vitamins, particularly those of the B group. Some examples

are pantothenic acid (Vitamin B5), which forms part of the important

coenzyme A; riboflavin (vitamin B2), incorporated into the molecules of

flavin-adenine di-nucleotide (FAD), and pyridoxal (vitamin B6), a cofactor

of ransaminases and decarboxylases.

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Enzymes have great specificity for their substrates and will frequently

not accept related molecules of a slightly different shape. This can be

explained by assuming that enzyme and substrate have a lock-and-key

interaction.

The RNA molecules in the nucleus synthesise different types of

proteins and thus enzymes (since enzymes are proteinous in nature). Since

the enzymes conrol all the metabolic processes, we can very safely say that

the enzyme of the nucleus ultimately governs all the metabolic processes of

the body.

The organelles important in connection with localization of various

enzymes and coenzymes in the nucleus are mitochondria, its matrix,

endoplasmic reticulum and microsomes.

Many enzymes associated with carbohydrates, fatty acids and nitrogen

metabolism are located within the mitochondrion. Enzymes of electron

transport and oxidative phosphorylation are also located in different areas of

this cell organelle.

The enzymes responsible for citric acid cycle and fatty acid oxidation

are located in the DNA, ribosomes and enzymes reqired for the biosythesis

of the proteins coded in the mitochondrial genome. The mitochondrion is

not, however, genetically autonomous, and the genes encoding most

mitochondrial proteins are present in nuclear DNA.

Table 1.3 gives the names of some of the important enzymes and their

location.

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Table 1.3 L Showing location of some of the important enzymes in

mitochondrion

Outer Membrane Intermediate Space Inner Membrane Matrix

Cytochrome b5 Adenylate kinase Cytochromes b, C,

C, a and a3

Pyrurate

dehydrogenase

complex (PDH)

Cytochrome b5

reductase

Sulfite oxidase NADH

dehydrogenase

Citrate synthase

Fatty acid CoA

synthetase

Nucleoside

diphosphokinase

Sccinate

dehydroenase

A conitase

FA elongation

system

Ubiquinone Socitrate

dehydrogenase

(ICD)

Phospholipase A Electron-

transferring flavo

Proteins (ETF)

-oxo-gltarate

dehydrogenase

Nucleoside

diphosphokinase

Vector ATP

synthetase (F0F1)

FA oxidation

system

-OH-butyrate

dehydrogenase

Ornithine

transcarbamoylase

Carnitine-Palmityl

transferases

Carbamoyl

phosphae synthetase

I

All ranslocases

The mitochondrion is specialized for the rapid oxidation of NADH

(reduced NAD) and FAD. H2 (reduced FAD) produced in the reactions of

glycolysis, the citric acid cycle and the oxidation of fatty acids. The energy

produced is trapped and stored as ATP, for future use of energy in the body.

A number of important enzymes are associated with the endoplasmic

reticulum of mammalian liver cells. These include the enzymes responsible

for the synthesis of sterol, triacyl glycerol (T-G), Phospolipids (PL) and the

enzymes involved in deloxification of drugs. Cytochrome P450 which

participates in drug hydroxylation reside in the ER.

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Lysosomes are cell organelles found in cells which contain packet of

enzymes. Lysosome word derived from Greek word "Gree", meaning lysis

(loosening). Discovered and described for the first time as a new organelle

by the Belgian Biochemist de Duve in 1955.

Essentially the enzymes abot 30 to 40, are hydrolytic in nature. They

can be grouped as follows :

(Table 1.4)

1. Proteolytic enzymes Cathepsins (Proteinase)

Collagenase

Elastase

2. Nucleic acid

hydrolyzing enzymes

Ribonucleases

Deoxyribonuleases

3. Lipid hydrolyzing anzymes Lipases

Phospholipases

Fatty acyl esterases

4. Carbohydrate splitting enzymes -glcosidase

- galactosidase

Hyaluronidase

Aryl sulfatase, etc.

5. Other enzymes Acid phosphatase

Catalase, etc.

As long as the lysosomal membrane is intact, the encapsulated

enzymes can act only locally. But when the membrane is ruptured, the

enzymes are released into the cytoplasm and can hydrolyze external

substrates (biopolymers)

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The outsides of the membranes of the endoplasmic reticulum contain

small granula, commonly known as microsomes or ribosomes.

In addition to the certain enzymes concerned with protein synthesis,

microsomes also possess steroid reductase (involved in metabolism of

cholesterol and steroid hormones), phosphatases hydroxylases, hydrolases,

glucuronyl transferase, and ATPase.

Golgi apparatus, mentioned earlier, acts as the store house of various

hormones and secretory enzymes which are released as such at the time of

requirement by the process of emeiocytosis.

1.4.3 DNA and RNA in Living Systems

Nucleic acids are macromolecules of the utmost biological

importance. All living organisms contain nucleic acids in the form of

deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Some viruses

contain only DNA, while others have only RNA.

DNA is the major store of genetic information. This information is

copied or transcribed into RNA molecules, the nucleotide sequences of

which contain the "code" for specific amino acid sequences. Proteins are

then synthesized in a process involving the translation of the RNA. The

series of events just outlined is often referred to as the central dogma of

molecular biology, and can be summarized in the form:

oteinPrRNADNA ntranslatioiontranscript

In higher cells, DNA is localized mainly in the nucleus, within the

chromosome. A small amount of DNA is present in the cytoplasm and

contained in mitochondria and chloroplasts. RNA is found both in the

nucleus, where it is synthesized, and in the cytoplasm, where the synthesis of

proteins takes place (Table 1.5).

27

Table 1.5 DNA and RNA-Structure, Reactions, and role in the cell

Deoxyribonucleic Acid Ribonucleic Acid

Localization Primarily in nucleus;

also in mitochndria and

chloroplasts

In cytoplasm,

nucleolus, and

chromosomes.

Pyrimidine bases Cytosine Cytosine

Thymine Uracil

Purine bases Adenine Adenine

Pentose Deoxyribose Ribose

Cytochemical reaction Feulgen Basophilic dyes with

ribonuclease treatment

Hydrolyzing enzyme Deoxyribonuclease

(DNase)

Ribonuclease (RNase)

Role in cell Genetic information Synthesis of proteins

3H Precursor

3H Thymidine

3H Uridine

Nucleic acids consist of a sugar moiety (pentose), nitrogenous bases

(purines and pyrimidines), and phosphoric acid.

A nucleic acid molecule is a linear polymer in which the monomers

(nucleotides) are linked to gather by means of phosphodiester "bridges" or

bonds. These bonds link the 3' carbon in the pentose of one nucleotide to the

5' carbon in the pentose of the adjacent nucleotide. Thus the backbone of a

nucleic acid consists of alternating phosphates and pentoses. The

nitrogenous bases are attached to the sugars of this backbone.

28

A mild hydrolysis cleaves the nucleic acid into component nucleotides

that result from the covalent bonding of a phosphate and a heterocyclic base

to the pentose.

Pentoses are of two types : ribose in RNA, and deoxyribose in DNA.

The only difference between these two sugars is that there is one less oxygen

atom in deoxyribose. A cytochemical reaction specific for the deoxyribose

moiety, called the Feulgen reaction, can be used to visualize DNA under the

microscope

The bases found in nucleic acids are also of two types : pyrimidines

and purines. Pyrimidines have a single hetorocyclic ring, whereas purines

have two fused rings. In DNA the pyrimidines are thymine (T) and cytosine

(C); the purines are adenine (A) and guanine (G). RNA contains uracil (U)

instead of thymine (Table. 1.5)

It is useful to remember that there are two main differences between

DNA and RNA: DNA has a deoxyribose and RNA a ribose moiety; DNA

contains thymine and RNA uracil. The difference in pyrimidine bases has

made it possible for cell biologists to use radioactive thymidine as a specific

DNA label, and radioactive uridine to label RNA in living cells.

Heterocyclic bases absorb ultraviolet light at a wavelength

(designated ) of 260 nm. A cell photographed at =260 nm, shows the

nucleolus (containing RNA), the chromatin, and the RNA-containing

regions of the cytoplasm, all of which absorb intensely. In fact, a simple way

of determining the concentration of nucleic acid in solution is to measure the

absorption at 260 nm.

29

The combination of a base plus a pentose, minus the phosphate,

constitutes a nucleoside. For example, adenine is a purine base; adenosine

(adenine+ ribose) is the corresponding nucleoside; while adenosine

monophosphate (AMP), adenosine diphosphate (ADP), and adenosine

triphosphate (ATP) are nucleotides (Fig. 1.5)

In addition to functioning as the building blocks of nucleic acids,

nucleotides are important because they are used to store and transfer

chemical energy. Figure 1.5 shows that the two terminal phosphate bonds of

ATP contain high energy. When these bonds are cleaved, the energy

released can be used to drive a variety of cellular reactions. The high energy

–P bond enables the cell to accumulate a large quantity of energy in a small

space and keep it ready for use when needed. In Figure 1.5 B it is possible to

appreciate how the energy stored in ATP can be used in different

biosynthetic pathways. Other nucleotides, such as cytosine triphosphate

(CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), also

have high energy bonds but the energy source for all of them is ultimately

derived from ATP.

DNA is present in living organisms as linear molecules of extremely

high molecular weight. E coli, for example, has a single circular DNA

molecule of 3,400,000 base pairs and has a total length of 1.4 mm. In higher

organisms the amount of DNA may be hundreds of times larger (700 times

in the case of man); for example, the DNA in a single human diploid cell, if

fully extended, would have a total length of 1.7m.

30

Fig. 1.5 : A, structure of ATP and its components. Note the presence of two

high energy phosphate bonds. B. channeling of phosphate bond energy by

ATP into specific biosynthetic routes. (Cortesy of A.L. Lehninger).

31

All the genetic information of a living organism is stored in the linear

sequence of the four bases. Therefore, a four-letter alphabet (A, T, G, C)

must code for the primary structure (i.e., the sequence of the 20 amino acids)

of all proteins. One of the most exciting discoveries in molecular biology

was the elucidation of this code. One prelude to this discovery, having direct

bearing on the understanding of DNA structure, was the finding that there

were predictable regularities in base content. Between 1949 and 1953

Chargaff studied the base composition of DNA in great detail. He found that

although the base composition varied from one species to another, in all

cases the amount of adenine was equal to the amount of thymine (A=T). The

number of cytosine and guanine was also found to be equal (C=G).

Consequently, the total quantity of purines equals the total quantity of

pyrimidines (i.e., A +G = C +T). On the other hand, the AT/GC ratio varies

considerably between species. For example in man the ratio is 1.52 while in

E. coli the ratio is 0.93.

Watson and Crick established the structure of the DNA molecule in

1953 on the basis of the x-ray diffraction studies of Wilkins and Franklin.

The model is formed of two right-handed helical polynucleotide chains that

are antiparallel and in which the bases are stacked perpendicularly. The two

chains are held together by hydrogen bonds between the base pairs. Only

two types of base pairs are possible within the double helix: AT and GC.

This explains the regularities observed in the molar ratios of bases. AT pairs

are held together by two hydrogen bonds, and GC pairs have three; for this

reason GC pairs are more stable. The double helix has a major and a minor

grove and completes one turn every 10 base pairs. A left-handed

conformation (Z-DNA) has been found.

32

The axial sequence of bases along one chain is a complement of the

other; DNA duplication takes place via a template mechanism, following the

unwinding of the two strands. The sequence of bases in a long polymer

provides an explanation for the immense number of combinations that carry

different genetic information.

The primary structure of RNA is similar to that of DNA, except for

the substitution of ribose for deoxyribose and uracil for thymine, as has

already been discussed (see Table 1.5) The base composition of RNA does

not follow Chargaff's rules, since RNA molecules consist of only one chain.

There are three major classes of RNA : messenger RNA (mRNA).

transfer RNA (tRNA), and ribosomal RNA (rRNA). All are involved in

protein synthesis – mRNA carries the genetic information for the sequence

of amino acids; tRNA identifies and transports amino acid molecules to the

ribosome, and rRNA represents 50% of the mass of ribosomes, the

organelles that provide a molecular scaffold for the chemical reactions of

polypeptide assembly.

Although each RNA molecule has only a single polynucleotide chain,

RNA is not a simple, smooth, linear structure. RNA molecules have

extensive regions of complementarily in which hydrogen bonds between A

and GC pairs are formed between different regions of the same molecule.

Figure 1.6 shows that as a result the molecule folds upon itself, forming

structures called hairpin loops. In the base-paired regions the RNA molecule

adopts a helical structure comparable to that of DNA. The compact structure

of RNA molecules folded upon themselves has important biological

consequences. For example, in bacteriophage MS2 a sequence that indicates

33

the starting site for one of its proteins (called the polymerase) is in a part of

the molecule inaccessible to the ribosomes, as shown in Figure 1.6 and can

only be expressed when additional factors unfold the molecule. The viroids

have no proteins and consist of a naked circular RNA molecule that can

produce diseases in plants

Fig. 1.6 : Part of the nucleotide sequence of the RNA baceriophage MS2,

a virus that infects E. coli, Note that the molecule folds back on itself,

forming hairpin loops.

1.4.4 Helix Coil Transition

As has been mentioned earlier in 1953, based on the x-ray diffraction

data of Wilkins and Franklin, Watson and Crick proposed a model for DNA

structure that provided an explanation for is regularities in base composition

and its biological properties, particularly its duplication in the cell. The

structure of DNA is shown in Figure 1.7 It is composed of two right-handed

helical polynucleotide chains that form a double helix around the same

central axis. The two strands are antiparallel, meaning that their 3', 5'

phosphodiester links run in opposite directions. The bases are stacked inside

the helix in a plane perpendicular to the helical axis.

34

Fig. 1.7: The DNA double helix. The phosphate-ribose backbones are

indicated as ribbons. The base pairs are flat structures stacked one on top of

another perpendicular to me long axis of DNA, and they are therefore

represented as horizontal lines in this side via.

The two strands are held together by hydrogen bonds established

between the two sugar moieties in the opposite strands, and only certain base

pairs can fit into the structure. The only two pairs that are possible are AT

and CG. It is important to note that two hydrogen bonds are formed between

A and T, and three are formed between C and G, and that therefore a CG

pair is more stable than an AT pair. In addition to hydrogen bonds,

hydrophobic interactions established between the stacked based are

important in maintaining the double helical structure.

35

The axial sequence of bases along one poly-nucleotide chain may vary

considerably, but on the other chain the sequence must be complementary,

as in the following example:

Because of this property, given an order of bases on one chain, the

other chain is exactly complementary. During DNA duplication the two

chains dissociate and each one serves as a template for the synthesis of a

new complementary chain. In this way two double-stranded DNA molecules

are produced, each having exactly the same molecular constitution.

Most of the DNA in a cell is right-handed and of the B-DNA form,

which is the most stable configuration described above. Some local regions,

however, can form a slightly different right-handed DNA called A-DNA.

Crystallographic studies on synthetic nucleotides consisting of alternating

purines and pyrimidines have shown that left-handed double helices can also

exist. This form has been called Z-DNA because of the zigzag array of the

phosphateribose backbone, as can be observed in the space-filling models

shown in Figure 1.8. In the Z-DNA configuration different parts of the

molecule are exposed and this may have biological consequences. In fact,

this alternate configuration suggests that DNA is a more flexible molecule

than was previously thought and that it can adopt in the genome a variety of

forms (Rich, 1980).

Because the structure of the DNA double helix is preserved by weak

interactions (i.e., hydrogen bonds and hydrophobic interactions established

between the stacked bases), it is possible to separate the two strands by

treatments involving heating, for example, or alkaline pH. This separation is

36

called melting or denaturation of DNA. Since the temperature required to

break the GC pairs (having three hydrogen bonds) is higher than that needed

to break the AT pairs (having two hydrogen bonds), the temperature at

which the DNA strands separate (the melting point) depends on the AT/GC

ratio.

If the DNA is cooled slowly after renaturation, the complementary

strands will base-pair in register, and the native (double helical)

conformation will be restored. This process is called renaturation or

annealing, and is a consequence of the base-pairing properties of

nucleotides.

Fig. 1.8 Two molecular forms of the DNA double helix, right-handed B-

DNA and left-handed Z-DNA. The heavy black line in each goes from

phosphate group to phosphate group, indicating a smooth right-handed helix

in B-DNA, but an irregular left-handed helix in Z-DNA.

(Courtesy of A. Rich)

37

Renaturation of DNA is a very useful tool in molecular biology. It

shows how DNA renaturation can be used to estimate the size (number of

nucleotides) of the genome of a given organism. When DNA is renatured

under standardized conditions, a large genome (e.g., calf) takes more time to

reanneal than a small genome (e.g., E. coli or bateriophage T4). This is

because the individual sequences take longer to find the correct partners (the

larger the genome, the more chances there are of incorrect molecular

collisions).

Renaturation studies led to the discovery of repeated sequences in

eukaryotic DNA. When certain DNA sequences are repeated many times,

the rate of renaturation will be much faster than for sequences present as

single copies. Some sequences (called satellite DNAs) can be repeated

millions of times in the genome.

Single-stranded DNA will also anneal to complementary RNA,

resulting in a hybrid molecule in which one strand is DNA and the other is

RNA. Molecular hybridization is a very powerful method for characterizing

RNAs because and RNA molecule will hybridize only to the DNA from

which it was transcribed.

Labeled probes can be made by using an RNA molecule (e.g., globin

messenger RNA). radio-active nucleosides and the enzyme reverse

transcriptase that copies a complementary DNA strand (cDNA). This cDNA

can then be used to localize the gene from which the RNA molecule arose.

Frthermore, using the modern methods of genetic engineering almost any

gene can now be cloned and used as a probe for hybridization.

38

Denaturation done under carefully controlled conditions may be used

for physical mapping of DNA in a technique known as partial denaturation

mapping. This technique is based on the fact that the regions rich in AT

separate more easily. Under the EM those regions are detected as single-

stranded loops, and the distance between the loops and the end of the DNA

molecule can be measured.

CHECK YOUR PROGRESS-1

Note : (1) Write your answers in the open space given below.

(2) Compare your answers with those given at the end of this unit.

(a) (i) Cell is the .......................... and ......................... unit of life.

(ii) The chief organelles of a cell are :

(a) .......................................

(b) .......................................

(c) .......................................

(d) .......................................

(e) .......................................

(f) .......................................

(iii) Nucleus controls all the ..................... of a cell. Its important

components are.

(a) .......................................

(b) .......................................

(c) .......................................

(d) .......................................

(e) .......................................

(f) .......................................

39

(iv) DNA .................................. and RNA ....................... of ..............

occurs in the nucleus.

(b) (i) Functions of proteins are related with ..................., .

..................., ......................., ....................... and ...................

(ii) Enzymes are ......................... The organelles important in

connection with localization of various enzymes are

..................., ................., .................. and ...................

(iii) All living organisms contain nucleic acid in the form of

............... and .................... DNA is he carrier of ...................

encoded in the ....................

(iv) Watson and Crick established the .................. Structure of

DNA, which is composed of ....................

1.5 CELL MEMBRANE AND TRANSPORT OF IONS

As has been mentioned earlier, the boundary of every cell, which

separates the cell contents from the outer environment, is known as cell

membrane. The membrane is believed to be formed by the ingredients of

the plasma and has selective permeability that mediate the flow of molecules

and ions into and out of the cell. They also contain molecules at their

surfaces that provide for cellular recognition and communication.

These membranes are composed of lipids, proteins and carbohydrates.

The relative content of these components – varies widely from one type of

membrane to another, but typically it contains 40% of the dry weight is

lipids, about 60% proteins and 1 to 10% carbohydrates. All membrane

carbohydrate is covalently attached to proteins or lipids.

40

(a) Lipids are the basic structural components of cell membranes. Lipid

molecules have a 'polar' or ionic head hence hydrophilic and the other end is

a 'nonpolar' and hydrophobic tail. Hence they are amphipathic. In

biomembranes the various types of lipids present are -

1. Fatty acids : These are the major components of most membrane

lipids. The nonpolar tails of most membrane lipids are long chain fatty acids

attached to polar head groups such as glyceroltriphosphate.

About 50% of the fatty acid groups are saturated i.e. they contain no

double bond. The most common saturated fatty acid groups in membrane

lipids in animals contain 16 to 18 carbon atoms. The other half of fatty acid

molecules contain one or more double bonds i.e. unsaturated or

polyunsaturated fatty acids. Oleic acid is the most abundant unsaturated fatty

acid in animal membrane lipids, others are Arachidonic acid, Linoleic and

Linolenic acids. The degree of unsaturation determines the fluidity of the

membranes.

2. Glycerophospholipids : They are another group of major components

of bio-membranes. Phosphatidyl ethanol amine (cephalin), phosphatidyl

choline (Lecithin) and phosphatidyl serine are among the most of common

glycerophospholipids.

3. Sphingolipids : They comprise another group of lipids found in

biological membranes specially in the tissues of nervous system. There are

three types of sphingolipids sphingomyelin, cerebrosides and gangliosides.

About 6 of the membrane lipids of grey matter cells in the brain are

gangliosides.

41

4. Cholesterol : Cholesterol is another common component of the bio-

membranes of animals but not of plants and prokaryotes. It is oriented with

its hydrophilic polar heads exposed to water and its hydrophobic fused ring

system and attached hydrocarbon groups buried in the interior. Cholesterol

helps to regulate fluidity of animal membranes.

(b) Proteins :

The different types of proteins present in membranes are –

1. Integral Membrane Proteins : (also called 'intrinsic' membrane

proteins) : These proteins are deeply embedded in the membrane.

Thus portions of these proteins are in Vander Waals contact with the

hydrophobic region of the membrane.

2. Peripheral Membrane Proteins (also called "extrinsic" proteins):

These may be weakly bound to the surface of the membrane by ionic

interations or by hydrogen bonds that form between the proteins and

the 'polar' heads of the membrane lipids. They may also interact with

integral membrane proteins. They can be removed without disrupting

the membrane.

3. Trans Membrane Proteins : Some of the integral proteins span the

whole breadth of the membrane and are called as "trans membrane

proteins". The hydrophobic side chains of the amino acids are

embedded in the hydrophobic central core of the membrane. These

proteins can serve as "receptors" for hormones, neurotransmitters,

tissue specific antigens, growth factors etc.

42

(c) Carbohydrates : Many membrane proteins are glycosylated, having

one or more covalently attached polysaccharides chains. The carbohydrate

coat is called the "glycocalyx". These chains may contain the mono-

saccharides D-galactose, D-mannose, L-fucose and the derivatives like N-

acetylglucosamine and N-acetyl-neuraminic acid.

They are attached to the proteins either by an N-glycosidic linkage

from N-acetylglucosamine to asparagine or by an O-glycosidic linkage from

N-acetyl galactosamine to serine or threonine.

The amino acid sequences around the carbohydrate attachment sites of

different proteins are often similar, presumably because they have to be

recognized by glycosylation of enzymes. The carbohydrate chains of may

glycoproteins show structural variation from one molecule to another, a

phenomenon known as "micro heterogeneity".

Electron micrographs of membranes of varios origin are remarkably

similar and show that the membrane consists of two dark banks each of 20Aº

thickness separated from a light band of about 35Aº as its thickness.

The dark bands are composed of proteins; On the other hand, the light

band is composed of phospholipids and cholesterol. The phospholipid

molecules in the light band are arranged in two rows in a way that the

phosphate containing ends, known as polar or hydrophilic ends point to the

outside while the non-polar or hydrophobic chains point to the inside (Fig.

1.2)

Lipid bilayers are oriented with their hydrophobic tails inside the

bilayer while hydrophilic 'polar' heads are in contact with the aqueous

solution on each side. Not all lipids can form bilayers. A lipid bilayer can

43

form only when the cross-sectional areas of the hydrophobic tail and

hydrophilic polar head are about equal. Glycerophospholipids and

sphingolipids fulfil this criteria and hence can form bilayer. The

lysophospholipids have only one fatty acyl group, it cannot form the bilayer

as the polar heads are too large, similarly cholesterol also cannot form

bilayers as the rigid fused ring systems and additional nonpolar tails are too

large.

The hydrophobic effect and the solvent entropy provide the driving

force for the formation of lipid bilayer. A lipid bilayer is about 6nm across

and this is so thin that it may be regarded as a two-dimensional flud. Lipid

molecules in a bilayer are highly oriented.

However, according to the fluid mosaic model of membrane structure

proposed by Singer and Nicholson in 1972, the membrane proteins, intrinsic

proteins (integral) deeply embedded and peripheral proteins loosely

attached, float in an environment of fluid phospholipid bilayers. It can be

compared like icebergs floating in sea water.

It has been shown that it is not only the integral proteins, the

phospholipids also undergo rapid redistribution in the plane of the

membrane. This diffusion within the plane of the membrane is termed

"translational disfusion". It can be quite rapid for a phospholipid molecule.

Within the plane of the membrane, one molecule of phospholipid can move

several micrometers per second. The phase changes, and thus the fluidity of

the membrane are highly dependant upon the lipid composition of the

membrane.

44

As the temperature increases, the hydrophobic side chains undergo a

transition from the ordered state which is more gel like or crystalline to a

disordered state, taking on a more liquid like or fluid arrangement. The

temperature at which the structure undergoes the transition from ordered to

disordered state is important.

The nature and function of these proteins are as follows :

1. Integral proteins : Two major integral proteins are found in red cells

membrane. They are :

(a) Glycophorin and

(b) Band-3-Protein.

(a) Glycophorins : Glycophorins are glycoproteins. It contains 60%

carbohydrates by weight. The oligosaccharides bound to glycophorin are

linked to serine, theronine and aspargine residues.

Red blood cells membrane contains about 6x105 gly cophorin

molecules. The polypeptide chain of glycophorin conains 131 amino acid

residues. A sequence of 23 hydrophobic amino acid residues lies within the

non-polar hydrocarbon phase of the phospholipid bilayer, tightly associated

with phospholipids and cholesterol. This 23 amino acid residue sequence has

an -helical conformation.

Function :

(i) Some of the oligosaccharides of glycophorin are the M and N

blood group antigens.

(ii) Other carbohydrates bound to glycophorin are sites through which

the influenza virus becomes attached to red blood cells.

45

(b) Band-3 Protein It is another major integral protein found in red cell

membrane. It is dimeric having molecular weight of 93,000. The polypeptide

chain of the dimer is thought to traverse the membrane about a dozen time.

Both the C and N terminals of band-3-protein are on the cytosolic side of the

membrane. The N-terminal residues extend into the cytosol and interact with

components of the cytoskeleton.

Functions : Band-3-protein plays an important role in the function of red

blood cells. As red blood cells flow through the capillaris of the lungs, they

exchange bicarbonate anious (HCO3-) produced by the reaction of CO2 and

H2O, for chloride (Cl-) ions. This exchange occurs by way of a channel in

band-3-protein, which forms a "Pore" through the membrane. Thus band-3-

protein is an example of a membrane transport protein.

2. Peripheral proteins : The inner face of the red blood cells membrane

is laced with a network of proteins called cytoskeletons that stabilizes the

membrane and is responsible for the biconcave shape of the cells:

The special peripheral membrane proteins participate in this stability

of red cells are Spectrin, Actin and Ankyrin and Band 4, 1 protein.

Spectrin : Spectrin consists of an -chain, having molecular weight

240,000 and a -chain having moleclar weight 220,000. It is a fibrous

protein in which the polypeptide chains are thought to coil around each other

to give an - dimer, 100 nm long and 5 nm in breadth.

Spectrin dimers are linked through short chains of actin molecules and

band 4, 1 proteins to form 2 2 tetramer.

46

Actin : In red blood cells and other non-muscle cells, actin is a component

of the cytoskeleton. An erythrocye contains 5 x 105 actin molecules. About

20 actin molecules polymerize to form short 'actin' filaments.

Ankyrin : The network of spectrin, actin and band 4, 1 protein forms the

skeleton of the red blood cell, but none of these proteins is attached directly

o the membrane. The network of proteins is instead attached to another

peripheral protein called "ankyrin".

Ankyrin has a molecular weight 200,000. The protein has 2 domains:

one binds to spectrin, and the other to the N-terminal region of band-3-

protein that extends into the cyloskeleton.

It is now known that the protein network can also be bound directly to

glycophorin (integral protein) or to band-3-protein.

1.5.2 Ion Transport through cell Membrane

An essential role of biomembranes is to allow movement of all

compounds necessary for the normal function of a cell across the membrane

barrier. These compounds include a vast array of substances like sugars,

amino acids, fatty acids, steroids, cations and anions These compounds must

enter or leave the cells in an orderly manner for normal functioning of the

cell.

Salts dissociated into anions (e.g., Cl-) and cations (e.g., Na

+ and K

+)

are important in maintaining osmotic pressure and the acid-base equilibrium

of the cell. Retention of ions produces an increase in osmotic pressure and

thus the entrance of water. Same the inorganic ions, such as magnesium, are

indispensable as cofactors in enzymatic activities; others, such as inorganic

47

phosphate, form adenosine triphospate (ATP), the chief supplier of chemical

energy for the living processes of the cell, through oxidative

phosphorylation.

The concentration of various ions in the intracellulate fluid differs

from that in the interstitial fluid. For example, the intracellular fluid has a

high concentration of K+ and Mg

++, while Na

+ and Cl

- are localized mainly

in the interstitial fluid. The dominant anion inside cells is phosphate; some

bicarbonate is also present.

Calcium ions are found in the circulating blood and in cells where

they play important regulatory roles. In bone they combine with phosphate

and carbonate ions o form a crystal line arrangement.

Phosphate occurs in the blood and tissue fluids as a free ion, but much

of the phosphate of the body is bound in the form of phospholipids,

nucleotides, phosphoproteins, and phosphorylated sugars. As primary

phosphate (H2PO4-) and secondary phosphate (HPO4

2-), phosphate

contributes to the acid-base equilibrium, thereby buffering the pH of the

blood and tissue fluids.

Other ions found in tissues are sulfate, carbonate, bicarbonate,

magnesium, and amino acids.

Certain mineral ions are also found as part of larger macromolecules.

For example, iron, bound by metal-carbon linkages, is found in hemoglobin,

ferritin, the cytochromes and some enzyme (such as catalase and cytochrome

oxidase). Traces of manganese, copper, cobalt, iodine, selenium, nickel,

molybdenum and zinc are indispensable for maintenance of normal cellular

48

activities. The importance of some of these elements in enzymes is indicated

in Table 1.6

TABLE 1.6 FUNCTIONS OF SOME INORGANIC IONS IN CELLS

Ions Function in

Fe++

or Fe+++

Mg++

Cu++

Zn ++

Mn++

Co++

Mo

Ca++

Hemoglobin, cytochromes, peroxidases

Chlorophyll, phosphatases

Tyrosinase, ascorbic acid oxidase

Carbonic anhydrase, peptidase, alcohol

dehydrogenase, Transcription Factor

IIIA

Peptidases

Peptidases

Nitrate reductase, xanthine oxidase

Calmodulin, actomyosin, ATPase

The following are three important mechanisms for transport of various

compounds across the bio-membrane

(a) Passive or simple diffusion (b) Facilitated diffusion, and (c) Active

transport.

Passive or simple diffusion : It depends on the concentration gradient

of a particular substance across the membrane. The solute passes from

higher concentration to lower concentration till equilibrium is reached. The

process neither require any "carrier protein" nor energy. It operates

unidirectionally.

49

Initial rate (v) at which a solute(s) diffuses across a phospholipid

bilayer is directly proportional to the concenration gradient across the

membrane ([S] out and inversely proportional to the thickness (t) of the

membrane Thus,

t

)in]s[out]s([DN

D is the diffusion coefficient', which is expressed in terms of area

divided by time. The diffusion of molecules across a bilayer is described by

"Permeability coefficient", which is equal coefficient (D) divided by the

thickness of the membrane (t).

Facilitated Diffusion : It is similar to passive diffusion in that solute

move along the concentration gradient. But it differ from passive diffusion

in that it requires a carrier or transport protein. Hence the rate of diffusion is

faster than protein", Hence the rate of diffusion is faster than simple

diffusion. The process does not require any energy and can operates

bidirectionally. Mechanism of facilitated diffusion has been explained by

ping-pong model.

(c) Active transport : Active transport occurs against a concentration

gradient and electrical gradient. Hence it requires energy. About 40% of the

total energy requirement in a cell is utilized for active transport system. It

requires the mediation of specific "carrier or transport proteins".

The transport systems can be classified as follows :

1. Uniport system : This system involves the transport of a single

solute molecule through the membrane.

50

2. Co-transport system : D-Glucose, D-Galactose and L-amino

acids are transported into the cells by Na+, dependent co-transport

system. Na+ is not allowed to accumulate in the cells and it is

pumped out by "sodium pump". It may be-

(i) Symport system : It is a co-transport system in which the

transporter carries the two solutes in the same direction across the

membrane. OR

(ii) Antiport system : It is a type of co-transport system in which two

solutes or ions are transported simultaneously in opposite

directions.

Example : chloride and bicarbonate ion exchange in lungs in red

blood cells.

The exocytosis also plays an important part in the transport

mechanism. Most cells release macromolecules to the exterior by the process

called exocytosis. This process is also involved in membrane remodelling

when the components synthesized in the Golgi apparatus are carried in

vesicles to the plasma membrane. The movement of the vesicle is carried out

by cytoplasmic contractile elements in the micro-tubular system.

Mechanism : The innermembrane of the vesicle fuses with the outer

plasma membrane, while cytoplasmic side of vesicle fuses with the

cyoplasmic side of plasma membrane. Thus, the contents of vesicles are

externalised. The process is also called "reverse pinocytosis". The process

induces a local and transient change in Ca++

concentration which triggers

exocytosis.

51

1.5.3 Thermodynamic Treatment of Membrane Transport.

In a resting state, there is no difference in electrical potential on either

side of the membrane and the membrane is said to be in an isopotential state.

Though the resting membrane is said to be in an isopotential state. Though

the resting membrane is in an isopotential state, the proximity of external

surface membrane contains a slight excess of cations over anions whereas

the proximity of inner surface membrane, a slight excess of anions over

cations due to ionic influx. Because of these slight changes in ionic

distribution, a voltage difference of -75 to 95 mV occurs at the two surfaces

of the membrane and is called resting membrane potential. The resting

potential is maintained by the active transport of ions against

electrochemical gradient by sodium/potassium pumps. During resting state,

the concentration of potassium ions is higher concentration of sodium ions

(142 mE/l) and that of sodium ions are lower (10 mE/L) inside the

membrane. The fluid outside the membrane has higher concentration of

sodium ions (142 mE/l) and lower concentration of potassium ions (5mE/l).

These differential ionic distributions are due to sodium and potassium

pumps by diffusion and active transport. Therefore the outside membrane

becomes positively charged and the inside membrane becomes negatively

charged because of the presence of higher concentrations of negatively

charged ions such as chlorides and proteins. In a resting state of the nerve,

there is an active transport of sodium ions from the axoplasm into the

interstitial fluid. This transport of sodium ions is known as sodium pump.

When two solutions containing diffusible and non-diffusible ions are

separated by a semipermeable membrane, the nondiffusible ions enhance the

diffusion of oppositely charged diffusible ions. The diffusion take place

towards nondiffusible ion containing side. This also reduces the diffusion of

52

like charged ions to that side. As a result, on the side which contains

nondiffusible ions, diffusible counterions are more concentrated while the

like charged diffusible ions concentrate more on the opposite side. This is

called as Gibbs-Donnan effect. However, the total number of cations and

anions is equal on both sides at equilibrium.

Let us assume there are two compartments A and B, which are

separated by a semi-permeable membrane.

The compartment A contains a solution of sodium proteinate in which

Na exists as Na+ and protein as Pr

- and to maintain elecrical neutrality

Na+ balances Pr

- Sodium salt of protein is a collidal solution, the

proteinate Pr- is colloidal and is not diffusible through the membrane.

The compartment B contains a solution of NaCl, in which both Na+

and Cl- are diffusible.

(90) Na+ Na

+ (90)

(90) Pr- Cl

- (90)

(A) (B)

membrane

According to Donnan effect, the non-diffusible ion or ions on one side

of the membrane influences the diffusion of diffusible ions, and both quality

and quantity of diffusible ions will be influenced.

In above situation, Na+ ions can diffuse either way, but Cl

- ions can

diffuse only to the left, i.e. to 'A' compartment containing non-diffusible Pr-,

whereas Pr-, cannot diffuse at all. After equilibrium is attained, the following

will be the situation in both compartments.

53

(120)Na+ Na

+ (60)

(90) Pr- Cl

- (60)

Cl-(30)

(A) (B)

membrane

In the side (A) : there will be more Na+, as Na

+ ions have to balance

now in addition to the existing non-diffusible Pr-, the newly entered Cl

- to

maintain electrical neutrality. On the other hand, in side (B) Na+ has to

balance only Cl- which are remaining, after diffusion to 'A' side. Hence, the

concentration of Na+ in side (A) will be greater than that of Na

+ in side (B)

(some Na+ will diffuse from B to A)

Na+ (A)> Na

+(B)

Thus, total ionic concentration in side (A) will be much greater than

side (B). The concentration of Cl- ions in (A) should be much less than in

side (B), i.e. Cl- (A) <Cl

-(B). On the other hand. Cl

- concentration in side (B)

will be > side (A) Cl- (B)>Cl

-(A).

At equilibrium, the product of diffusible ions on either side of the

membrane will be equal.

Thus, the Donnan effect has brought the following changes in above

example.

1. On the side in which non-diffusible ion is present, there is

accumulation of oppositely charged diffusible ions, i.e. Na+.

2. In the other side of the membrane, the non-diffusible ions have

made the accumulation of diffusible ions of the same, i.e. Cl-.

54

3. The total concentration of all the ions will be greater in which the

non-diffusible Pr- is present leading to osmotic imbalance between

the two sides.

1.5.4 Nerve Conduction

In most animals, coordination is effected by means of neurons or

nerve cells, which are capable of transmitting or conducting the impulses in

the form of electrical wave through specific pathways. According to Prosser,

the sum of total physical and chemical reactions in the conduction of a wave

of physiological activity along the nerve fibre is called an impulse. When the

nerve is subjected to external stimuli, it is excited and sets up a wave of

impulse, which travels along the nerve fibres.

The action potential occurs in a nerve when it is stimulated by a

stimulus, which may be mechanical, electrical or chemical. The biochemical

processes associated with nerve impulses are very poorly understood in

detail : however, two phenomena are clearly of major importance : (1) the

movement of sodium and potassium ions through the nerve cell membrane,

and (2) the production, release, and hydrolysis of acetylcholine.

We normally think of an electrical current as a flow of electrons;

however, we should recall that in an aqueous solution electron transport

occurs with the movement of ions. It should not be difficult, then, to realize

that the movement of ions in and out of a nerve fiber can give rise to

electrical impulses. The control of the movement of ions through the nerve

cell membrane is another matter. Nerve cells, like all animal cells, have

lower Na+ and higher K

+ concentrations than the extracellular fluid. This

situation is associated with a difference in electrical potential across the cell

membrane. The maintenance of this nonequilibrium situation requires that

55

work be continually performed at the expense of the usual metabolic energy.

The mechanisms are nknown. Physiologists often speak of a hypothetical

device for the elimination of Na+ from cells, but it should be realized that

any such mechanism must be based on chemical processes.

On applying a stimulus to a non-myelinated nerve, the membrane of

the nerve fibre becomes depolarized and the sodium pump stops. The

permeability of the membrane for the sodium ions increases so that the

sodium ions from outside the membrane tend to move into the axoplasm.

This causes a change in the polarization of the membrane and the interior of

the fibre becomes positive while the outside becomes negative. This even is

just opposite of the resting potential and is called depolarization or reversal

potential. The prevalence of electropositivity inside and electronegaivity

outside the excited nerve fibre is called action potential. The flow of sodium

ions from the original depolarized area proceeds further in both directions.

Thus more and more sodium ions enter the axoplasm until the whole interior

of the nerve fibre becomes electronegative. This movement of sodium ions

as an electric current, which spreads along the membrane, is called

depolarization wave or nerve impulse. As soon as the depolarization wave

spreads completely over the entire length of the fibre, sodium ions cannot

enter further into the axoplasm. But a large quantity of potassium ions

diffuses through the membrane into the axoplasm. Now the sodium ions

begin to diffuse out simultaneously with the resultant electronegativity

inside the membrane and electropositivity outside. This process occurs at the

same point of the nerve fibre in which depolarization has started and extends

in both directions. This is called repolarization during which the sodium

pump starts once again.

56

The whole process of depolarization and repolarization is completed

within a small fraction of a second and thus the nerve fibre can conduct

impulses very rapidly.

In vertebrates, the efficiency of impulse conduction is increased by

myelin sheath, which surrounds the axon, acting as an effective insulator. In

myelinated nerves also the action potential arises in the same manner as in

the non-myelinated nerves but only at the nodes of Ranvier, which lack the

sheath. Here, here the action potential actually jumps from one node to

another and the process is called saltatory conduction. In myelinated nerves,

the current spreads outwards from the node so that the action potential

reaches the next mode from its origin faster than the impulse, which would

travel along the same disance in a non-myelinated nerve fibre. Therefore the

velocity of conduction depends on the diameter of the nerve fibres.

Whatever the precise nature of the mechanisms involved in the

transmission of an impulse along a nerve fiber, there remains another

problem- the transmission of the impulse from one nerve cell to another or to

a receptor such as a muscle fiber. Apparently this is accomplished by the

release from the nerve ending of a compound which stimulates other cells

with which it may come in contact. Basically this resembles hormone action,

but the effect is a very local one and essentially provides only for cell-to-cell

information transfer. Nevertheless the compounds involved are sometimes

called neurohormones. Although other compounds may serve similar roles,

two compounds are of general significance. These are acetylcholine and

norepinephrine. These compounds are released at the endings of the

"cholinergic nerves" and the "adrenergic nerves," respectively.

57

Acetylcholine is the ester of choline with acetic acid. The compound

is apparently formed by nerve cells from choline and acetyl-CoA and stored

in small bundles near the nerve endings. With the arrival of an electrical

impulse, acetylcholine is released into the small space between the nerve

terminus and the surrounding cells. Upon contacting a cell membrane, the

compound apparently combines with a protein of the membrane in much the

manner that a substrate or a competitive inhibitor combines with an enzyme.

This combination alters the permeability of the cell membrane to small ions,

and presumably the subsequent changes in metabolic activity of the receptor

cell follow automatically.

After the transmission of the impulse in this way, conditions must be

restored to the initial state. This is accomplished with the hydrolysis of

acetylcholine by acetylcholinesterase, an enzyme associated with nerve

tissue. The reaction is given below :

H3C

N ―CH2―CH2―O―C―CH3+H2O inesteraseacetylchol

H3C | ||

N ` O

acetylcholine

H3C

N ―CH2―CH2OH + H―O―C― CH3

H3C | ||

N ` O

acetic acid

As the free acetylcholine around the nerve ending is hydrolyzed, the

bound acetylcholine with which it is in eqilibrium is released and also

58

hydrolyzed. The cell is then ready for another stimulus. In the nerve ending,

acetylcholine will also be resynthesized from choline and acetyl-CoA.

It has also been suggested that the hydrolysis and reformation of

acetylcholine within the nerve is associated with the changes in permeability

occurring with the nerve impulse.

Thus, in synapses, the impulse transmission is mediated by a

neurotransmitter substance (acetyl choline). When a nerve impulse reaches

the synaptic knob, it depolarizes the presynaptic membrane and causes the

permeability of the membrane to calcium ions. Now the calcium ions enter

into the knob and cause the fusion of synaptic vesicles with the presynaptic

membrane. The vesicles release their contents (neurotransmitter substance)

into the synaptic cleft by exocytosis. Then they return to the cytoplams for

refilling. The released acetylcholine diffuses across the synaptic cleft,

attaches to a specific receptor site on the postsynaptic membrane and causes

a change in the shape of the receptor site. This in turn results in opening of

ion channels in the postsynaptic membrane. Thus ion channels are opened in

response to binding of acetylcholine to receptor proteins. As a result, sodium

ions which enter through the postsynaptic membrane causes depolarization

of the membrane.This excites the cell to develop action potential (nerve

impulse). The acetylcholine is immediately removed from the synaptic cleft

by the enzyme acetyl cholinesterase present on the postsynaptic membrane.

The neuromuscular junction is a specialized synapse found between a

motor neuron and skeletal muscle fibres. It includes both motor end plate

and the synaptic knob of the neuron. The fine branches of neuron terminate

in shallow cavities of sarcolemma (cell surface membrane of the muscle

59

fibre), which has many deep folds containing receptors for acetylcholine in

the cavities. On stimulation, the synaptic knobs release acetylcholine by the

same mechanism as in synapses Change in structure of receptor sites on the

folds of sarcolemma (postsynaptic membrane) increases the permeability of

the sarcolemma to sodium and potassium ions resulting in a local

depolarization called end-plate potential. This potential causes an action

potential, which is transmitted along the sarcolemma to the muscle fibre

through the T system of the muscle fibre.

CHECK YOUR PROGRESS-2

Note : (1) Write your answers in the space given below.

(2) Compare your answers with those given at the end of the unit.

(a) (i) Cell-membranes are composed of ......................, ..................

and ........................

(ii) An essential role of biomembranes is to allow .................... of

all ..................... necessary for the .................................... of a

cell across the membrane ........................

(iii0 The three important mechanism for transport of various

compounds across the bio-membrane are :

(a) .................................

(b) .................................

(c) .................................

(iv) In resting state of the nerve, there is active ...............................

of ............................. from the axoplasm into

...................................... This transport of ........................... is

known as ..........................

60

(b) (i) In animals coordination is effected by means of ................... or

........................ which are capable of ................. or conducting

the ...................... in the form of ....................... through specific

............................

(ii) The biochemical processes associated with nerve impulses may

be–

(a) ........................................

and (b) .........................................

(iii) The neuro-muscular junction is a .............................. found

between a ........................... and ..........................

1.6 LET US SUM UP

By going through this unit you would have achieved the objectives

stated at the start of the unit. Let us recall what we have discussed so for :

Cell is the structural and functional unit of life. All cells arise from

other cells. The genetic information required during the maintenance

of existing cells and the production of new cells passes from one

generation to the other next generation. The chemical reactions of an

organism that is its metabolism, both anabolism and catabolism, takes

place in the cells.

Eukaryotic cells contain many membrane – bounded organelles that

carryout specific cellular processes. Chief organelles are :

(1) Cell membrane (2) Nucleus, (3) Mitochondria, (4) Endoplasmic

Reticulum, (5) Lysosomes and (6) Centrioles.

61

The membrane is believed to be formed by the ingredients of the

plasma (cytoplasm). It separates the cell contents from the outer

environment.

Nucleus is the heaviest particulate component of the cell. It lies

approximately in the middle of the cell. Its important components are :

(i) Nuclear envelope, (ii) Nuclerpore and complexes, (iii) Chromatin

(iv) Chromosomes (v) Nucleolus and (vI) Nucleoplasm.

Chromosomes are composed of nucleoprotein molecules, the DNA

molecules act as prosthetic group. DNA replication and RNA

transcription of DNA occur in the nucleus. Transcription is the first

step in the expression of genetic information and is the major

metabolic activity of the nucleus.

Mitochondria are the largest particulate components of the cytoplasm

and contribute about 35 percent of the total protein of the liver. They

are bounded by two concentric membranes that have markedly

different properties and biological functions.

Proteins are highly complex, natural compounds, composed of a large

number of different - aminoacids, and are the chief constituents of

protoplasm in all the living cells. In addition to the use of proteins as

food, they are of great importance. The important biological functions

of proteins include:

(i) as biological structural material (viz collagen),

(ii) as biological catalysts (i.e. enzymes),

(iii) as antibodies and

62

(iv) as nucleoproteins (i.e. important consequents of genes).

Enzymes are biological catalysts. Enzymes are the largest and most

specialised class of protein molecules. The represent one of the most

important products of the gene contained in the DNA molecule.

Many enzymes require the presence of a cofactor for their activities.

This may be a metal or a prosthetic group in the case of conjugated

proteins.

Nucleic acids are macromolecules of the utmost biological

importance, present in all living organisms in the form of DNA and

RNA. DNA is the major store of generic information. This

information is copied or transcripted into RNA molemolecules, the

nucleotide sequences of which contain the 'Code' for specific amino

acid sequences, Proteins are then synthesized in a process involving

the translation of the RNA.

All the gentic information of a living organism is stored in the linear

sequence of the four bases. There fore, a four-letter alphabet (A, T.G,

C) must code for must code for the primary structure of all proteins

(i.e. the sequence of the 20 amino acids).

There are three major classes of RNA : messenger RNA (mRNA –

carries the genetic information for the sequence of amino acid),

transfer RNA (t RNA- identifies and transports amino acid molecules

to the ribosome) and ribosomal RNA (r-RNA- represent 50% of the

mass of ribosomes).

63

The structure of DNA is composed of two right handed helical

polynucleotide chains that form a double helix around the same

central axis. The double helix structure is preserved by weak

interactions i.e. hydrogen bonds and it is possible to separate the two

strands by heating or by treatment with alkaline pH.

Single stranded DNA will also anneal to complementary RNA,

resulting in a hybrid molecule in which one strand is DNA and the

other is RNA.

Cell membrane is the boundary of every cell, which separate the cell

contents from the outer environment. These membranes are composed

of lipids, proteins and carbohydrates. An essential role of bio-

membranes is to allow movement of all compounds necessary for the

normal function of a cell across the membrane barrier.

The important mechanisms for transport of various compounds across

the biomembrane are :

(i) Passive or simple diffusion,

(ii) facilitated diffusion and

(iii) Active transport

Thermodynamic treatment of membrane transport involves 'sodium

pump' and 'Gibbs.Donnan' effect.

In a resting state there is no difference in electrical potential on either

side of the membrane and the membrane is said to be in an

isopotential state. In this state of the nerve, there is an active transport

64

of sodium ions from the axoplasm into the interstitial fluid. This

transport of sodium ions is known as sodium pump.

According to Donnan effect, the non-diffusible ion or ions on one side

of the membrane influences the diffusion of diffusible ions, and both

quality and quantity of diffusible ions will be influenced.

In most animals coordination is effected by means of neurons or nerve

cells, which are capable of transmitting or conducting the impulses in

the form of electrical wave through specific pathways.

The action potential occurs in a nerve when it is stimulated by a

stimulus, which may be mechanical electrical or chemical. The

biochemical processes associated with nerve impulses may be:

(i) The movement of sodium and potassium ions through the nerve

cell membrane, and

(ii) The production, release and hydrolysis of acetyl choline.

1.7 CHECK YOUR PROGRESS : THE KEY

1. (a) (i) Structural

Functional

(ii) (a) Cell membrane

(b) Nucleus

(c) Mitochondria

(d) Endoplasmic Reticulum

(e) Lysosomes

(f) Cenrioles

65

(iii) activities

(a) Nuclear envelope

(b) Nuclear pore complexes

(c) Chromatin

(d) Chromosomes

(e) Nucleolus

(f) Nucleoplasm

(iv) replication

transcription

DNA

(b) (i) hormones, collagen, enzymes, haemoglobin, nucleoproteins.

(ii) biological catalysts

mitochondria, its matrix, endoplasmic reticulum

microsomes.

(iii) DNA

RNA

genetic information

genes

(iv) double helix

polynucleotide chains

2. (a) (i) lipids

proteins

carbohydrates

(ii) movement

compounds

function

barrier

66

(iii) (a) Passive or simple diffusion

(b) Facilitated diffusion

(c) Active transport

(iv) transport

sodium ion

interstitial fluid

sodium ion

sodium pump

(b) (i) neurons

nerve cells

transmitting

impulse

electrical wave

pathways

(ii) (a) The movement of sodium and potassium ions through the

nerve membrane.

and (b) production, release and hydrolysis of acetyl choline.

(iii) specialised synapse

motor neuron

skeletal muscle fibres.

UNIT- 2 METAL IONS IN BIOLOCIAL SYSTEMS

Structures

2.1 Introduction

2.2 Objectives

2.3 Essential and Trace Metals

2.4 Sodium/Potassium Pump

67

2.5 Transport and Storage of Oxygen

2.5.1 Heme Proteins and Oxygen uptake

2.5.2 Structure and Function of Hemoglobin

2.5.3 Myoglobin

2.5.4 Hemoglobin and Haemerythrin

2.5.5 Model Synthetic Complexes of Iron, Cobalt and Copper

2.6 Let Us Sum Up

2.7 Check Your Progress: The Key

2.1 INTRODUCTION

Metal compounds are closely related to the life process. Among these

compounds are hemoglobin, chlorophyll, numerous hematin enzymes (e. g.

catalase and cytochrome oxidase), metal activated enzymes, vitamin B12

and

those vital but poorly understood complexes which play an important role in

the metabolism of the metallic ions.

Hemoglobin, which allows vertibrates to remove oxygen from the air

is an iron (Fe2+

) complex, while chlorophyll1, which plays a central role in

photosynthesis of plants, is a magnesium complex. Besides, compounds

having similar structures (heme-proteins) are used in life processes for a

variety of purpose, most of which are catalytic in nature. These molecules

are capable of aiding the transfer of both electrons and simple molecules

from one chemical species to another.

Many enzymes and metabolites are metal complexes and perhaps

most of the enzyme function only as metal complexes.

The proper understanding of the aspects of biochemical processes is

68

limited due to the complex structures of the biochemical molecules. Eichron

underlined the participation of coordination compounds in almost every

phase of biological activity. All these processes involve one or more

enzymes, which need metallic ions for their activity and have been shown to

function as metal complexes1.10

described below:

(a) In the natural process of bond-formation and bond rupture, such as (i)

during cleavage of peptide bonds (i.e. metabolic decomposition of

protein into amino acids), the enzymes involved are end peptidases,

such as chymotrysin (Ca2+

-complex), exopeptidase (bivalent metal

complexes), dipetidases such as glycylglycine dipeptidase (Co2+

-

complex) or glycyl-L-leucine dipeptidase (Zn2+

/Mg2+

-complex)

aminopeptidases (Mg2+

/Mn2+

-complex) and carboxypeptidases (Mg2+

-

complexes), (ii) during carboxylation and decarboxylation reactions

(i.e. the addition and removal of carbon dioxide), such as conversion

of oxalosuccinic acid to -ketoglutaric acid and of -ketoglutaric

acid to succinic acid, the carboxylase enzymes involved are

magnesium and manganese complexes, while carbonic anhydrase is a

Zn(II) complex, (iii) during phophorylation reactions (i.e. synthesis or

destruction of phosphate bonds, the energy source of biochemical

reactions), such as conversion of ATP to ADP, by phosphorylase

(Mg2+

-complex), or the activity of contractile protein, actomyosin

(Mg2+

-complex) or the biological activity of insulin (Zn2+

-complex),

the metal-enzyme complexes functions as catalyst. Similarly, during

the other condensation and cleavage reactions, such as coenzyme-A

(Mg2+

/Ca2+

-complex) with oxalo-acetic acid enol to form citric acid or

the functions of enolase (Mg2+

-complex) during the dehydration of D-

69

2, phosphoglyceric acid to phosphoenol pyruvate depends on the

metal-enzyme-complex catalysis.

(b) The exchange of functional groups, such as transamination (i.e.

transfer of amino groups from amino acids to keto-acids) involve

pyridoxal (Cu2+

/Fe2+

-complex) coenzyme.

(c) The blocking of functional groups of polyfunctional molecules, e.g.

blocking of ornithine portion during degradation of arginine to

ornithine and urea, is catalysed by arginase (Mn2+

-complex).

(d) In influencing the stereochemical configuration (i.e. coordination as a

means to provide orientation to the substrate for the reaction to occur),

e.g. in the activity of porphyrins (Fe2+

-complex) or hydrolase (i.e.

prolidase, a Mn2+

-complex) in peptide hydrolysis.

(e) In oxidation-reduction reactions which involve oxidoreductase

enzymes, such as phenol oxidase, tyrosine, lacease, ascorbic oxidase

(ali Cu2+

-complexes), hematin (Fe-complex), cytochromes (Fe-

complex), etc.

(f) In storage and transfer reactions, such as transportation of oxygen, i.e.

functioning of hemoglobin (Fe-complex), hemocynin (Cu2+

-complex)

and cyanocobalamin (Co2+

-complex), and

(g) In transmission of energy, e.g. functioning of chlorophyll (Mg2+

-

complex) during photosynthesis.

Thus, metal ions play important part in different fields of biological

activity. The important ions in this respect are: Na+, K

+, Ca

++, Mg

++ like

70

strong electropositive metals, which play important part in ionic equilibrium

and transition metal ions like Cu++

, co++

, Fe++

, Zn++

, Mn++

, Mo4+

etc. which

play important part in enzyme activities.

2.2 OBJECTIVES

The main aim of this unit is to discuss importance of the participation

of metal ion in various biological processes. After going through this unit

you would be able to:

underling the various essential and trace metals, important with

respect to biological activity.

discuss the mechanism of Na+/K

+ pump.

describe the mechanism of transport and storage of oxygen and

discuss the part played by hemoglobin, myoglobin and haemerythrin

in the transportation of oxygen.

2.3 ESSENTIAL AND TRACE METALS

It is observed that there are at least 29 different types of element in

our body. Organic components such as carbohydrates, proteins, and lipids

form about 90% of the solid matter and mainly consist of C, H, O and N.

The metals of the body are divided in two major groups:

(1) Nutritionally important minerals or principal metals. The daily

requirement of these is > 100 mg. The deficiency of these can prove

fatal. These include Na, K, Ca, Mg etc. They are also called macro

metals.

71

(2) Trace metals which are essential. The requirement in less than 100 mg

per day. Deficiency can lead to serious disorders. They include Cr, Co,

Cu, Fe, Mn, Mo, Se, Zn etc.

2.3.1 ESSENTIAL METALS

Essential metals are defined as those elements which are essential to

maintain the normal living state of a tissue or the whole of the body. These

elements depending upon their absolute amounts in the body, are further

divided into two groups macro elements and bulk metals, e.g.

(a) Calcium:

Calcium is an important mineral mainly found in bone and teeth. The

total calcium of the body is 25-35 mols (100 g-170 g). About 99% of it is

found in bones. It exists as carbonate or phosphate of calcium. About 0.5%

in soft tissue and 0.1% in ECF. The normal level of plasma calcium is 9-11

mg/dl. The calcium in plasma is of 3 types namely,

(a) ionized calcium (diffusible),

(b) protein bound calcium and

(c) complexed calcium, it is probably complexed with organic acids.

About 40% of total calcium is in ionized form. Albumin is the major

protein with which calcium is bound. All the three forms of calcium

in plasma remain in equilibrium with each other. Ionized calcium is

physiologically active from of calcium.

It is widely distributed in food substance such as milk, cheese, egg-

yolk, beans, lentils, nuts, figs. cabbage.

72

Calcium is taken in the diet principally as calcium phosphate,

carbonate and tartarate. Unlike Na and K which are readily absorbed, the

absorption of Ca is rather incomplete. About 40% of average daily dietary

intake of Ca is absorbed from the gut. Calcium is absorbed mainly from the

duodenum and first half of jejunum against electrical and concentration

gradients.

Two mechanisms have been proposed for absorption of calcium by

gut mucosa.

(a) Simple diffusion

(b) An "active" transport process involving energy and Ca++

pump. Both

the processes require 1, 25-dihydroxy-D3 (calcitriol) which regulates

the synthesis of Ca-binding proteins and transport and also a Ca++

-

dependant ATPase.

Factors Affecting Absorption

Various factors which influence the absorption of calcium are

discussed below :

1. pH of intestinal milieu: An acidic pH favours calcium absorption

because the Ca-salts, particularly PO4 and carbonates are quite soluble

in acid solutions.

In an alkaline medium, the absorption of calcium is lowered due to the

formation of insoluble tricalcium PO4.

2. Composition of the diet:

(i) High protein diet: A high protein diet favours absorption, 15% of

dietary Ca is absorbed. If the protein content is low, only 5% may be

absorbed.

73

Reason: Amino acids increase the solubility of Ca-salts and thus its

absorption. Lysine and Arginine obtained from basic proteins cause

maximal absorption of Ca.

(ii) Fatty acids: In malabsorption syndrome, fatty acids are not

absorbed properly. Fatty acids produce insoluble calcium soaps

which are excreted in faeces, thus, decreasing the Ca absorption.

(iii) Sugar and Organic Acids: Organic acids produced by microbial

fermentation of sugars in the gut, increases the solubility of Ca-salts

and increases their absorption. Citric acid also may increase the

absorption of calcium.

(iv) Phytic acid: Cereals contain phytic acid (inositol hexaphosphate)

which forms insoluble Ca-salts and decreases the absorption of Ca.

(v) Oxalates: Oxalates present in vegetable like cabbage and spinach

forms insoluble calcium oxalates which are excreted in the faeces,

thus lowering the calcium absorption.

(vi) Fibres: Presence of excess of fibres in the diet interferes with the

absorption of calcium.

(vii) Minerals:

Phosphates: Excess of phosphates lower calcium absorption.

Magnesium: High content of magnesium in the diet decreases

absorption of calcium.

Ca: P ratio : A ratio of good Ca to P not more than 2:1 and not

less than 1:2 (ideal 1:1) is necessary for optimal absorption of

calcium.

74

Fe in diet: Food Fe may form insoluble ferric phosphates. These

indirectly increases the Ca:P ratio in the gut beyond the range of

optimal absorption.

(vi) Vitamin D: Promotes Ca absorption.

3. State of health of the individual and aging:

(i) A healthy adult absorbs about 40% of dietary calcium.

(ii) Above the age of 60 years, there is a gradual decline in the intestinal

absorption of Ca.

(iii) In sprue syndrome, the intestinal absorption of calcium suffers due

to formation of Ca-soap with FA which are excreted in faeces.

4. Hormonal:

(i) PTH (Parathormone): PTH directly cannot increase the calcium

absorption. But PTH stimulates "1 - hydroxylase" enzyme in the

kidney and increases the synthesis of 1, 25-(OH)2-D3 (calcitriol)

which enhances calcium absorption.

(ii) Calcitonin: Calcitonin directly cannot affect Ca-absorption.

Increased calcitonin level inhibits "1 - hydroxylase" enzyme, thus

decreasing synthesis of calcitriol and Ca-absorption.

(iii) Glucocorticoids: Diminishes intestinal transport of calcium.

Calcium is secreted into the gut as a normal constituent or bile and

intestinal fluids. Faecal output of calcium could exceed intestinal absorption

on situations where the diet contains high levels of phytates or other

sequestrating substances. Under normal circumstances the faeces are not an

important excretion route for calcium.

75

Regulation: Kidneys filter about 250 mMole of Ca++

every day, some 95%

of which is reabsorbed by the tubules. The major portion of this filtered Ca+2

is taken up by proximal tubule without hormonal regulation. A fine

adjustment to the amount reabsorbed occurs in distal tubules under the

influence of PTH (PTH-uptake). Plasma level of ionized calcium conc. is the

principal regulator of PTH secretion by a simple negative feedback

mechanism. A threshold level of magnesium is required for PTH release.

Hypermagnaesemia inhibits PTH secretion. PTH secretion is also subject to

negative feedback by the vit D metabolite 1.25 (OH)2D3 PTH rapidly

stimulates osteoblast activity, the increased bone resorption causing an

increase in plasma Ca+2

and PO4-

Vit D3 plays a permissive role for this

effect. PTH stimulates more slowly (days) osteoblast activity. PTH via c-

AMP increases the distal nephron reabsorption of calcium and decreases that

of PO4 in the proximal tubule. In doing so, PTH increases the tubular

synthesis and excretion of c-AMP. PTH also stimulates the enzyme complex

that converts 25, OH D3 to 1, 25 (OH)2D3, thereby increasing calcium uptake

from the gut. Hypercalcemi stimulates calcitonin and katacalcin release

while hypocalcemia has inhibitory effect. Calcitonin strongly inhibits

osteoblastic bone resorption. However the role of calcitonin in calcium

regulation is controversial. Thyroid hormones, ACTH prostaglandins have

some effect on Ca level of plasma.

Functions

(i) Calcification of Bones and Teeth: The process of bone formation and

teeth formation is known an calcification which is a continues process

for bones. Osteoblasts secrete an enzyme alkaline phosphatase which

can hydrolyze certain Phosphoric esters.

76

(ii) Calcium plays a role in blood coagulation by producing substance for

thromboplastic activity of blood.

(iii) Calcium has a role in neuromuscular transmission.

(iv) Calcium ions are needed for excitability of nerves.

(v) Calcium plays role in muscle contraction.

(vi) Normal excitability of heart is Ca ion dependent.

(vii) It plays role as secondary or tertiary messenger in hormone action.

(viii) It plays role in permeability of gap junctions.

(b) Magnesium:

Magnesium is the fourth most abundant and important cation in

humans. It is extremely essential for life and is present as intracellular ion in

all living cells and tissues.

Sources: Magnesium is widely distributed in vegetable, found in

prophyrin group of chlorophyll of vegetables and also found in almost all

animal tissues. Other important sources are cereals, beans, green vegetables

potatoes, almonds and dairy products e.g. cheese.

Distribution: Total body magnesium is approximately 2400 mEq.

Approximately 2/3 occurs in bones, 1% in E.C. fluid and remainder in soft

tissues:

(i) Plasma level: 1.5 to mEq/L, which is rigorously maintained within

normal limits. 15% of total body Magnesium is exchangeable with

tissues but there is a wide variation. Muscles contain 20% of

exchangeable Mg and bonds only 2%. Hyperthyroidism markedly

increases the amount of exchangeable Mg. whereas it is reverse in

hypothyroidism.

77

(ii) Blood: Magnesium exists in blood partly bound to proteins. Under

conditions of physiological pH roughly 1/3 is 'protein-bound', the

remainder 2/3 is ionic.

(iii) C.S. Fluid: Concentration of Mg in C.S. Fluid is ½ as high as in

plasma.

Average daily intake in humans is 250-300 mg. much of which is

obtained from green vegetables where Mg is found in porphyrin group of

chlorophyll. Roughly 1/3 of dietary Mg is absorbed; the remainder is

passively excreted in faeces. Absorption takes place primarily in small

bowel, beginning within hour after ingestion and continues at a steady rate

for 2 to 8 hours, by that time 80% of total absorption has taken place.

Factros Affecting Absorption

(i) Size of Mg load : Absorption is double when normal dietary Mg

requirement is doubled and vice versa.

(ii) Dietary calcium: Increases absorption in calcium deficient diets.

Decreased absorption occurs in presence of excess of Ca. A common

transport mechanism from intestinal tract for both Ca and Mg

suggested.

(iii) Motility and mucosal state: This also affects absorption. In hurried

bowel absorption is decreased. Absorption decreases in damaged

mucosal state.

(iv) Vit-D: helps in increased absorption.

(v) Parathormone: increases absorption.

(vi) Growth hormone: increases absorption.

78

(vii) Other factors:

High protein intake and Neomycin therapy increase absorption.

Fatty acids, phytates and phosphates decrease absorption.

Excretion: Magnesium is lost from the body in faeces, sweat and urine. 60

to 80% or orally taken Mg is lost in faeces.

Sweat loss: Currently it is drawing attention; 0.75 mEq of Mg is lost daily in

perspiration in normal health with normal diet. Loss is much increased with

visible frank sweating.

Urine: Regulation of Mg balance is principally dependent on renal handing

of the ion. In a normal healthy adult with normal diet 3 to 14 mEq. are

excreted daily.

Factors Affecting Renal Excretion:

(i) Calcium intake: Increased dietary calcium produced increased

excretion of Mg.

(ii) Parathormone (PTH): diminishes excretion.

(iii) Antidiuretic hormone (ADH): increases Mg excretion.

(iv) Growth hormone (G.H): also in increases excretion of Mg.

(v) Aldosterone: increases excretion.

(vi) Thyroid hormones: 80% greater excretion in hyperthyroidism.

(vii) Alcohol ingestion: oral ingestion of as little as 1.0 ml of 95%, alcohol

per kg. Increases urinary excretion 2 to 3 fold. The increased excretion

partially accounts for Mg-deficiency in chronic alcoholics with

Delirium tremens.

79

(viii)Administration of acidifying substance (NH4Cl) is followed by

increased urinary elimination of Mg.

Functions:

1. Role in Enzyme Action: Mg is involved as a cofactor and as an

activator to wide spectrum of enzyme actions. It is essential for

peptidases, ribonuclease, glycolytic enzymes and co-carboxylation

reactions.

2. Neuromuscular Irritability: Mg exerts an effect on neuromuscular

irritability similar to that of Ca++

, high levels depress nerve conduction

and low levels may produce tetany (hypomagnasemic tetany).

3. As constituent of Bones and Teeth: About 70% of body magnesium is

present as appetites in bones, dental enamel and dentin.

Plasma Mg in Diseases:

(a) Hypermagnaesemia: Raised values have been reported in:

Uncontrolled Diabetes Mellitus,

Adrenocortical insufficiency,

Hypothyrodisim,

Advanced renal failure, and

Acute renal failure.

(b) Hypomagnaesmia: Low values are observed in:

Malabsorption syndrome and kwashiorkor,

Prolonged gastric suctions,

Hyperthyrodisim,

Portal cirrhosis,

Prolonged use of diuretics,

80

Chronic alcoholism,

Delirium tremens,

Renal diseases,

Primary aldosteronism

Magnesium Deficiency:

In man, 'overt' magnesium deficiency rarely occurs.

1. In Animals: In cattles, two types:

Unsupplemented whole milk (in calves).

Endemic disease: called as Grass staggers (or Grass tetany). Cattles

grazing in fields fertilized with Nitrates. Condition occurs due to high

NH3 content of diet. Absorption of Mg is impaired by the formation of

insoluble ammonium-Mg-phosphates.

2. In Humans: Experimentally induced prolonged Mg-depletion reported

in two patients (reported by Shils). Both were fed Mg-deficient

synthetic diets: one for 274 days and another for 414 days. In both,

plasma Mg fell slowly over several months.

(c) Sodium:

Sodium is the chief electrolyte which is found in large conc. in

extracellular fluid compartment. Approx, body distribution of sodium is as

follows:

Total m Mol Conc. in Mol/L

Total body 3150 -

Intracellular 250 10

Extracellular 2900 140

Plasma 400 140

81

The sodium is found in the body mainly associated with chloride as

NaCl and NaHCO3.

Sources: Sodium is widely distributed in food material more in animal

sources than plants. However, major source is table-salt used in cooking or

seasoning. It is also found in cheese, butter, khoa. Daily requirement of

sodium is as follows:

1-3.5 g of Na is required daily for adults.

Infants need 0.1-0.5 g and

Children 0.3-2.5 g daily.

Functions:

(i) Sodium maintains crystalloid osmotic pressure of extracellular fluids

and helps in retaining water in E.C.F.

(ii) Along with other cations Na+ is also involved in neuromuscular

irritability which is given:

Neuromuscular irritability = ][][][

][][

HMgCa

NaK

(iii) Acid base balance: Na+-H

+ exchange in renal tubule to acidify urine.

(iv) Maintenance of viscosity of blood: The salts of Na with globulins are

soluble and further Na+ and K

+ both regulate in maintaining the degree

of hydration of the plasma proteins.

(v) Role in resting membrane potential: Plasma membrane has a poor Na+

permeability and passive Na+ inflow through it. Na-pump keeps Na

+

conc. far higher outside than inside. This separation of charges is called

82

polarization of the membrane. It creates a potential difference of-70 to

95 mill volts across the membrane and is called as resting membrane

potential.

(vi) Role in Action Potential: A local depolarization of never or muscle

fiber is observed in stimulation. This rapidly increase its permeability to

Na+ causing considerable transmembrane influx of Na

+ down its inward

conc. gradient.

(d) Potassium:

Potassium is the major intracellular cation. It is widely distributed to

the body fluids and tissues as follows:

Whole blood : 200 mg/dl

Plasma : 20 mg/dl

Cells : 440 mg / 100 g

Muscle tissue : 250-400 mg / 100 g

Nerve-tissue : 530 mg / 100 g

It is widely distributed in the vegetable foods. Average amount of 4g

of potassium is percent in the potassium is easily absorbed.

As soon as it is absorbed, potassium enters the cells. It is excreted in

the urine. The amount of potassium excretion increases, when there is an

excessive dietary intake of sodium. Average normal human body contains

3.6 moles of potassium. The conc. entration of intracellular K+ is 150mEq/L

which is roughly equal to the conc. of sodium outside the cell. The normal

conc. of plasma potassium is 3.5-5 mEq/L. The Na+- K

+ATPase or sodium

pump maintains this concentration gradient. Potassium is also excreted in

gastrointestinal tract, saliva, gastric juice, bile, pancreatic and intestinal

83

juices. This fact becomes clinically important if these secretions are lost in

large amounts. Potassium is continuously filtered by the glomeruli of the

kidney and reabsorbed by the cells of proximal convoluted tubules.

Potassium (and hydrogen) ions are also secreted in distal tubule in exchange

for sodium.

Functions:

Many functions of potassium and sodium are carried out in

coordination with each other and are common. These functions have already

been described under sodium briefly:

(i) It influences the muscular activity.

(ii) Involved in acid-base balance.

(iii) It has an important role in cardiac function.

(iv) Certain enzymes such as pyruvate kinase require K+ as cofactor.

(v) Involved in neuromuscular irritability and nerve conduction process.

2.3.2 Trace Metals

Trace metals are those metals which are required in very small

amounts, almost in micrograms or nenograms, by the body. These are also

called oligometals and include iron, copper, cobalt, zinc, manganese,

molybdenum and selenium.

(a) Iron

Iron is an important element for all animal beings. The amount of iron

required by a body depends upon various factors e.g. age, sex, weight and

conditions are circumstances. It is required for the development of cells and

synthesis of hemoglobin. Maximum amount of iron is required by a kid

84

during first two years, because the growth rate is quite faster during this

period:

(i) Child (1-2 years) - 10 to 45 mg

(ii) Child (3-4 years) - 15 mg

(iii) Child (4-10 years) - 10 mg

(iv) Youth (Male) (11 to 18 years) - 18 mg

(v) Youth (Male) (after 19 years) - 10 mg

(vi) Youth (Male) (20-50 years) - 18 mg

(vii)Man (Male) (above 10 years) - 10 mg

An adult male requires approximately 10 mg/day and adult female 20

mg/day.

Dietary sources of iron are:

I. Exogenous: Foods rich in iron include:

a. Animal Sources: Meat, fish, liver, spleen, red marrow are very rich

sources (2.0 to 6.0 mg/100 gm). Also found in shellfish.

b. Vegetable Sources: Cereals (2.0 to 8.0 mg/100 gm) are the major

rich source. Legumes, molasses, nuts, amaranth leaves. Dates are

other good sources.

II. Endogenous: Iron (fe) is utilized from ferritin of RE system and

intestinal mucosal cells. Fe obtained from "effete" red cells are also

reutilized.

Absorption of iron and factors regulating absorption:

Around 10 to 20 mg of Fe is taken in the diet and only about 10% is

absorbed. The greatest need of iron is during infancy and adolescence.

85

The only mechanism by which total body stores of iron is regulated is

at the level of absorption. Garnic proposed a "mucosal block theory" for iron

absorption.

Mucosal Block Theory:

1. Soluble inorganic salts of iron are easily absorbed from the small

intestine. HCl present in gastric juice liberates free Fe3+

from non-heme

proteins. Vitamin C and glutathione in diet reduce Fe3+

to Fe2+

, which is

less polymerizable and more soluble form of iron. Vitamin C and

aminoacids can form iron-ascorbate and iron-amino acid chelates which

are readily absorbed. Heme is absorbed as such.

2. Gatroferrin, a glycoprotein in gastric juice is believed to bind iron and

facilitate its uptake in duodenum and jejunum.

3. The absorption of iron from intestinal lumen into mucosal cells takes

place as Fe2+

.

4. Events in intestinal mucosal cells:

In the mucosal cell cytoplasm, there is a carrier called intracellular

iron carrier (I.I.C.), Fe3+

iron is oxidized again in mucosal cells to Fe3+

form principally by ceruloplasmin (Ferroxidase I) and also to some

extent by ferroxidase II, both are Cu-containing enzymes.

Intracellular iron carrier delivers a fixed amount of iron to

mitochondria. It also transfers certain amount of Fe3+

to "apoferritin",

which is synthesized by mucosal cells, to form the storage form

"ferritin".

86

I.I.C. transfers some iron across the serosal cell membrane to a

plasma 1 -globulin, called apotransferrin to form transferrin. Iron is

carried in transferrin as Fe3+

.

The I.I.C. holds Fe3+

in either protein bound or chelated forms which

represent the "carrier-iron pool" in the intestinal mucosal cells.

Presence of sufficient amount of Fe in "carrier-iron pool". Keeps the

I.I.C. nearly or totally saturated and consequently reduces further iron

absorption. This theory advanced by Garnic is known as "mucosal

block theory", which regulates the iron absorption form the gut.

Others Factors:

(a) Source of Fe has marked effect on absorption:

Heme iron which comes mainly from animal products and is from

haemoglobin and myoglobin, is efficiently absorbed (about 20 to

30%).

Non-heme iron, which is present in plants, though ingested in larger

amount than heme iron, are inefficiently absorbed (only 1 to 5%).

(b) The absorption of non-heme iron is influenced by the:

Composition of the diet.

pH of the intestinal milieu, and

State of health of the individual.

1. Composition of the Diet: The composition of the diet exerts a

profound effect on non-heme iron absorption.

Dietary factors that increase iron absorption are the presence of

vitamin C (ascorbic acid), glutathione, and some form of meat, fish or

87

poultry (all contain an unknown "meat factor").

Foods that inhibit non-heme iron absorption to some extent are:

tea (diminishes absorption by > 60%).

coffee (reduced absorption by > 35%).

phytates, found in corn, soya products, grains and bran (producing

insoluble complex).

oxalates found in spinach and chocolates.

some dietary fibres may also bind the iron or decrease

gastrointestinal transit time.

2. pH of Intestinal Milieu:

HCl secreted in gastric juice liberates Fe3+

from non-heme iron and

serves to increase solubility of dietary non-heme iron.

pH of duodenum is most conducive for absorption. Rate of absorption

further decreases down the intestines as the pH becomes more

alkaline.

At high alkaline pH, the ingested iron is precipitated.

3. State of Health of the Individual:

Healthy adults absorb about 5 to 10% of dietary iron, which is approx.

imately 1 to 2 mg of iron.

Iron-deficient adults absorb 10 to 20% of the dietary iron equivalent

to 3 to 6 mg of Fe.

Iron Transport and Utilization: Transport of iron (Fe) throughout the body

is accomplished with a specific protein called transferrin. Transferrin is a

88

non-heme iron binding glycoproteins. Apotransferrin is the apoenzyme and

Fe is its prosthetic group. It has a molecular weight of 70,000 and it can bind

with two atoms of iron in the ferric state (Fe+++

) synergistically in presence

of HCO-3 ion. It exists in plasma as 1 globulin and is the true carrier of iron.

In plasma, transferrin is saturated only to the extent of 30% to 33% with

iron. Prior to binding to transferrin, Fe++

(ous) iron has to be oxidized to

Fe+++

(ic) form. Ceruloplasmin and ferroxidase II are required for this

conversion.

Function of Transferrin:

Major function of transferrin is transport of iron to R.E. cells, bone

marrow to reach the immature red blood cells. Specific receptors are

available on cells surface. Transferrin is internalized by receptor mediated

endocytosis. Within the target cells, iron is released and apotransferrin is

recycled to form new transferrin molecules.

Transferrin transports Fe from the GI tract to the bone-marrow for Hb

synthesis and to all other cells as required. Transferrin can transport a

maximum of two atoms of iron as Fe3+

per molecule. Normally, in

plasmalserum transferrin is about 33% saturated with Fe. As discussed

above, cell surface specific receptors are available for the iron-transferrin

complex. Tissues having high uptake, e.g. liver, have a larger number of

receptors present. The number of receptors decreases when a person is

replete with iron and increases with depletion. Iron is transported to bone

marrow where it is required for Hb synthesis. Fe2+

is incorporated in

protoporphyrin IX with the help of the enzyme "ferrochelatase".

89

Iron is also transported into cells where it is used for both oxidative

phosphorylation and as an enzyme and as enzyme cofactor. ,

A small amount of Fe is released each day from 'effete' red cells,

which are destroyed by phagocytes, but this released Fe2+

is recycled into

new Hb in the erythroblasts. A small amount of released Fe is also stored as

ferritin. The turnover of iron in an adult in 24 hours has been calculated to

be 35 to 40 mg. Plasma "transferrin iron pool" is in equilibrium with the iron

in storage forms ferritin and haemosiderin. Ferritin in storage form of Fe

occurs in reticuloendothelial system (RES), viz liver, spleen and bone

marrow and also in intestinal mucosal cells. When Fe is mobilized from

ferritin, the storage form, the sequence is as follows:

First call: from ferritin of RE system (Liver, spleen and bone

marrow)

Second call: from ferritin of intestinal mucosal cells.

Thirdly: absorbed iron from intestines.

Before Fe is released from ferritin to blood, Fe3+

of ferritin is first

reduced to Fe2+

.

Distribution of iron in the body is recorded in Table 2.1

TABLE 2.1: DISTRIBUTION OF IRON IN THE BODY

Protein/Enzyme Iron content

(in mg)

% of

total

1. Haemoproteins

Haemoglobin

Myoglobin

Heme enzyme Catalase and

peroxidase

25000

400

2-3

60-70

5-10

1

90

2. Organo-Iron-compounds

Cytochromes

4-5

1

3. Storage Iron

Ferritin

Haemosiderin (non heme protein)

300-700

10-15

4. Transferrin (non heme protein) 6-8 1

5. Iron requiring enzymes

Fp, Fe- Nonheme enzymes

Other-dehydrogenases

Nonheme enzymes

-

-

1

1

Functions:

Although the amount of total iron in the body is small, it performs the

following vital functions :

1. Oxygen carriage in blood: Iron is an essential constituent of

hemoglobin, which carries oxygen from lungs to other tissues.

2. Oxygen supply to muscles: The muscles store oxygen in combination

with myoglobin which also contains iorn.

3. Relation with tissue oxidation: Iron forms an integral part of all the

cytochromes and certain other enzymes, such as catalase and

peroxidaes. Cytochromes catalyze biological oxidation and provide

energy. It has also been found that iron forms a part of prosthetic group

of some of the flavoproteins and is thus involved in electron transfer.

91

4. In the development of normal R.B.C.: The development of R.B.C. is

completed by passing through a number of stages. One of these stages

requires iron for synthesis of hemoglobin which starts at this stage. If

iron is not available at this stage, the synthesis of hemoglobin will be

hampered leading to development of abnormal erythrocytes. Thus iron

is required in sufficient amounts for the normal development of

erythrocytes.

5. Relation with cell nucleus: The chromation material of nucleus

contains iron which possible takes an essential part in metabolic

oxidation taking place in nucleus.

6. Relation with oxidation in nerve cells: Iron is also present in nissl

granules of the neurons. These granles are seen only in the resting state

of neurone and disappear in the active state indicating the fact iron

plays an important role probably in the metabolic oxidation.

(b) Cobalt

Cobalt forms an integral part of vitamin B-12

and is required as a

constituent of this vitamin.

Sources and Requirement: Normal average diet contains about 5 to 8 g

of cobalt which is far more than the recommended daily allowance (1 to 2

g of vitamin B12 contains approximately 0.045 to 0.09 g of cobalt).

Main source: Foods from animal source. Not present in vegetables.

Absorption and Excretion: About 70 to 80% of the dietary cobalt is

absorbed readily from the intestine. Isotopic studies have shown that about

65% of the ingested cobalt is excreted almost completely through the

92

kidney. Cobalt is stored mainly in the liver being the principal storage site,

only trace amount present in other tissues.

Functions:

1. Role in Formation of Cobamide Enzyme: In formation of cobamide

coenzyme (Ademosyl co-enzyme), cobalt of B12 undergoes successive

reduction in a series of steps catalyzed by the enzyme "B12 reducates",

which requires NADH and FAD.

2. Bone Marrow Function:

Cobalt is required to maintain normal bone marrow function and

required for development and maturation of red blood cells. A

deficiency of cobalt results in decreased B12 supply which produces

nutritional macrocytic anemia.

Excess of cobalt results in overproduction of red blood cells causing

polycythaemia. The polycythaemic effect may be due to inhibition of

certain respiratory enzymes viz. cytochrome oxidase, succinate

dehydrogenase etc. leading to relative anoxia.

3. Role as Cofactor: Cobalt may act as a cofactor for enzyme like

glycyl-glycine dipeptidase of intestinal juice.

Cobalt Deficiently: In ruminants, but not in other species, cobalt deficiency

results in anorexia, fatty liver, macrocytic anemia, wasting and

haemosiderosis of spleen.

93

(c) Manganese

Manganese is also an essential trace element and required by the

body. The average diet can provide approximately 3 to 4 mg of manganese

which is obtained principally from cereals, vegetables, fruits, nuts and tea.

From animal source: liver and kidneys are rich source and can supply

sufficient Mn++

to meet the daily requirement.

Absorption: About 3 to 4% of dietary Mn++

is absorbed. Dietary Ca and P

have been found to reduce Mn++

absorption.

Distribution: The total amount of this trace element in our body has been

estimated to be approximately 15 mg average (Range 10 to 18 mg) and is

found concentrated mainly in the kidneys and liver.

Blood level: Blood contains about 4 to 20 g manganese, per 100 ml. It is

present mainly in red blood cells in combination with several porphyrins and

is transported in the plasma in combination with a 1 globulin called as '

transmanganin. Because of its presence in plasma in protein bound form

very little of it is excreted in urine.

Functions

1. Role in Enzyme Action :

Acts either as a 'cofactor' or as an activator of many enzymes like

arginase, isocitrate dehydrogenate, (ICD), Cholinesterase, lipoprotein

lipase, enolase, lucineaminopeptidase in intestine, phosphot-

ransferases and 5-oxo-prolinase of kidneys, and small intestine and

many others.

94

Manganese and magnesium may replace one another in case of some

of the enzymes.

Mitochondrial form of superoxide dismutase contains Mn++

in its

prosthetic group unlike the cytosol form of the enzyme which contains

Cu and Zn.

Another mitochondrial enzyme, the ATP-dependant tetrameric ligase

called pyruvate carboxylase contains Mn++

, and also the vitamin

Biotin in its prosthetic group, which is involved in ''CO2-fixation

reaction". A similar enzyme is Acetyl CoA carboxylase which also

contains Mn++

and biotin.

Mn++

may be associated with mitochondrial respiratory chain

enzymes. :

Manganese also acts as a cofactor of all hydirolases &decarboxylases.

2. Role in Animal Reproduction: In animals, Mn++

deficiency-has been

shown to produce sterility in cattles disturbances of estrous cycles,

resorptin of foetus and sterility in cows, and degeneration of testes as

well as inability to feed the offspring in rats

3. Role in Bone Formation: Mn++

plays a part in the synthesis or

deposition of Mucopolysaccharides (MPS) in the cartilaginous

matrices of long bones. Mn-deficiency causes significant lowering in

the content of chondroition SO4. Abnormal bone formation due to Mn

deficiency may lead to perosis (slipping of gastroenemius tendon) and

bone deformities in chicks

95

4. Role in Carbohydrate Metabolism: Mn++

is reported to influence

carbohydrate metabolism by affecting the peripheral utilization and

their conversion to MPS. In Mn deficiency, pancreatic hypoplasia,

associated with 'diabetic type' of G.T.T has been reported

5. Role in Porphyrin Synthesis: Some porphyrins of RB cells contain

Mn++

, Manganese also helps in porphyrin synthesis by participating in

ALA synthetaze activity. Hb-synthesis appears to be depressed in

Mn-deficient rats.

6. Role in Fat Metabolism: Manganese has been reported also to exhibit

"lipotropic effect" and it stimulates F.A. synthesis and cholesterol

synthesis.

7. Role in Proteoglycan Synthesis: Manganese also participates in

glycoprotein and proteoglycan synthesis.

(d) Copper

Adult humans contain 100 to 150 mg of copper, out of which

approximately 65 mg is found in muscles, 23 mg in bones und 18 mg in

liver. Foetal liver contains approximately ten times more copper than adult

liver.

It occurs as:

erythrocuprein (in red blood cells),

hepatocuprein (in liver) and

cerebrocuprein (in brain).

Erythrocuprein is a colourless protein containing 2 atoms of Cu per

molecule. Molecular weight approx. 33,000.

96

Source : Average diet provides 2 to 4 mg/ day in the form of meat, shellfish,

legumes, nuts and cereals. Milk and milk- products are poor sources.

Absorption: Primarily absorbed from the duodenum. About 32% of the

dietary Cu can be absorbed. Phytates, Zinc, Mo, Cd, Ag, Hg and high

amount of Vit C inhibit Cu absorption.

Absorption of Cu from GI tract requires a specific mechanism because

of highly insoluble nature of Cu++

ions. An unidentified low molecular

weight substance from human saliva and gastric juice complexes with Cu++

to keep it soluble at pH of intestinal fluid. In the intestinal mucosal cells, Cu

is associated with low molecular weight metal binding protein called as

metallo-thionein.

Plasma: After absorption Cu enters plasma, where it is bound to amino

acids, particularly histidine and to serum albumin at a single strong binding

site. In less than an hour, the recently absorbed Cu is removed from the

circulation by liver.

Role of Liver in Copper Absorption: Liver processes absorbed Cu through

two routes:

1. Cu is excreted in the bile into the GI tract from which it is not reabsorbed.

In fact, copper homeostasis is maintained almost exclusively by biliary

excretion, the higher the dose of the Cu more it is excreted in faeces.

Normally, human urine contains only traces of Cu.

2. Second route: Incorporation as an integral part of Caeruloplasm, a

glycoprotein synthesized exclusively by liver.

97

Serum Copper: Serum Cu level is approximately 90 %g (average). In red

blood cells; 93 to 115 g /100 ml. During pregnancy, the serum levels of Cu

rises steadily and reaches peak level at the time of parturition. Cu of red

blood cells, however, remains almost constant.

Serum Cu is present in two distinct forms:

Direct reacting Cu: which is loosely bound to albumin.

Approximately 4% present in this form. So-called as it reacts directly

with diethyldithiocarbamate.

Bound form: which remains bound to -globulin fraction of the

serum, called "Ceruloplasmin" (as stated above). About 96% of serum

Cu is found in combination with Ceruloplasmin.

Requirements:

Infants and children; 0.05 mg Cu/kg body wt. per day.

Adult requirement is approximately 2.5 mg/ day.

Ordinary diets consumed daily contain about 2.5 to 5.0 mg Cu.

Functions

1. Rote in Enzyme Action: Cu forms integral part of certain enzymes

e.g. some of cytochromes, cytochrome oxidase tyrosinase,

Monoamine oxidase (MAU), Lysyl oxidase Catalase, Ascorbic acid

oxidase, uricase and superoxide dismutase. These enzymes contain

about 550 g of Cu per gram of enzyme protein.

Superoxide dismutase: A colourless dimeric enzyme having mol.

wt = 32,000, present in cytosol of mammalian liver, nerve and red cells and

contain 2 Cu++

and 2 Zn++

per molecule.

98

Function of Superoxide dismutase: Changes superoxide radicals, formed

by univalent reduction of O2 in tissues hydrogen peroxide (H2O2)

O2 + O2

+ 2H

+ O2 + H2O2

There is another "mitochondrial" form of 'superoxide dismutase' enzyme

which is a different protein with Mn" instead of Cu++

and Zn++

as its

prosthetic group.

2. Role of Cu++

in Fe Metabolism:

Cu helps in the utilization of Fe for Hb synthesis in the body. It is

believed that 'caeruloplasmin', a blue-Cu protein complex of blood

plasma, functions as serum Ferro-oxidase, catalyzes the oxidation

of Fe++

to Fe+++

. This helps in the incorporation of Fe in

"transferrin" to facilitate mobilization and utilization of Fe.

Facilitatory role of Cu++

in iron absorption also.

A yellow copper-protein called serum ferro-oxidase II or non

ceruloplasmin fero-oxidase may also participate in the oxidation of

Fe++

in human plasma.

3. Role in Maturation of Elastin: Copper helps to form insoluble elastin

fibres by cross-linking soluble proelastin chains through the oxidation

of some Lysine side chains of the latter into aldehydes. Proelastin rises

significantly in copper deficient animals.

4. Role in Bone and Myelin Sheath of Nerves: Copper has been reported

to help in the formation of bones and maintenance of myelin sheaths of

nerve-fibres.

99

5. Role in Haemocyanin: One of the copper protein "thaemocyanin"

found in blood of certain invertebrates functions as Hb in the storage

and transport of O2.

(e) Zinc

Sources:

(a) Animal sources: Good sources of zinc are liver, milk and dairy

products, eggs.

(b) Vegetables sources: Good vegetable sources are unmilled cereals,

legumes, pulses, oil seeds, yeast cells, tables (spinach, lettuce).

Distribution: An adult man weighing 70 kg approximately 1.4 to 2.3 gm of

zinc in the body distributed in different parts of the body as follows :-

High (70 to 86 mg/100 gm): in skin, and prostate

Average (15 to 25 mg per 100 gm): in bones and teeth.

Low (2.3 to 5.5 mg/100 gm): in kidneys, muscles, heart, pancreas and

spleen and

Very low (1.4 to 1.5 mg/100 gm): in brain and lungs.

Absorption and Excretion:

Only a small percentage of dietary Zinc is absorbed and the

absorption occurs mainly from duodenum and ileum.

Zinc-binding factor: It has been reported and claimed that a low

molecular weight zinc-binding factor is secreted by the pancreas,

which forms complex with zinc and helps in its absorption.

High amounts of dietary calcium, phosphates and phytic acid have

been found to interfere with zinc absorption.

100

Loss in faeces and urine: In a normal healthy adult human, approximately

9.0 mg of zinc is lost in the faeces and about 0.5 mg is lost in the urine and

0.5 mg is retained in the body.

Sweat: Trace amount is lost in sweat.

Blood level: Whole blood contains about 650 to 680 g of zinc per 100 ml.

It is present in red blood cells mainly in carbonic anhydrase enzyme

molecules and W.B. cells as other zinc-protein complexes.

Plasma : Plasma contains approximately 120 to 140 g /100 ml of plasma,

mostly in combination with serum albumin.

Requirements: As a result of balance studies, the requirement for normal

health has been recommended as 0.3 mg zinc/per kg body wt. Adult men and

women require about 15 to 20 mg. Pregnant and lactating women the

requirement is 25 mg and for infants and children, it is 3 to 15 mg.

Functions :

1. Role in Enzyme Action: Zinc forms an integral part of several

enzymes (metallo-enzymes) in the body. Important zinc containing enzymes

are:

Supeoxide dismutase: the enzyme is present in cytosol of brain cells,

liver cells and blood cells. It is a Cu-Zn protein complex with two

Zn++

per molecule of the enzyme. .

Carbonic anhydrase: molecular weight is = 30,000. It is present in red

blood cells, parietal cells and renal tubular epithelial cells and

contains one Zn++

per molecule of the enzyme.

Lucine ammo peptidase (LAP): of intestinal juice.

101

Carboxy peptidase 'A': of pancreatic juice.

List of other enzymes is given in table 2.2

Table 2.2 : Examples of other zinc containing enzymes are :

Alcohol dehydrogenase of mammalian liver and yeast cells

Retinine reducates of retina

Alkaline phosphates enzyme

Glutamate dehydrogenase involved in transdeamination

Lactate dehydrogenase which brings about reversible reaction P.A to

LA.

DNA and RNA polymerase

-ALA dehydratase

2. Role in Vitamin A Metabolism: Zn++

has been claimed to stimulate

the release of vitamin A from liver into the blood and thus increases its

plasma level and its utilization in rhodopsin synthesis. In addition, Zn++

containing metallo-enzyme "retinene reducase" participates in the

regeneration of rhodopsin in the eye during dark adaptation after

illumination with light.

3. Role in Insulin Secretion: Protamine zinc-insulin and globin zinc

insulin contain Zn++

for its functioning. Rise of blood glucose after glucose

administration to a normal animal increases the release of Insulin

simultaneously lowers the zinc content of pancreas specially of -cells of

Islets of Langerhans. Zinc content of pancreas also have been found to

diminish in Diabetes mellitus. The above facts indicate the participation of

zinc in storage and secretion of Insulin.

102

4. Role in Growth and Reproduction:

(i) Prasad et. al. have shown that zinc deficiency lead to 'dwarfism' and

'hypogonadism'. In such dwarfs, zinc concentration in plasma, red cells,

hairs, urine and faeces was found to be less than control subjects. Pubic

hairs disappear do not grow. However, zinc caused improved growth and

appearance of pubic hair. Growth retardation and gonadal hypo-function in

these subjects were also related to zinc deficiency.

(ii) Zinc deficiency also lowers spermatogenesis in males and menstrual

cycles are disturbed in females.

(III) Zinc deficiency due to phytate-rich diet may cause poor body growth,

failure of full reproductive maturity and hypogonadism in humans.

5. Role in Wound Healing: Zinc is necessary for wound healing. Zinc

has been found to accumulate in granulation tissues and in and around the

healing wounds. Zinc deficiency delays wound healing. Thus zinc plays a

vital role in wound healing.

6. Role in Biosynthesis of Mononucleotides: The biosynthesis of

mono-nudeotides and their incorporation into the nucleic acids has been

found to be impaired in zinc deficiency. Ribonuclease activity has been

reported to be higher in zinc deficiency.

(f) Selenium

Selenium was found to prevent liver cell necrosis, which was

discovered by Schwartz and Flotz in 1957. Since then a wide variety of

animal diseases have been shown to respond to selenium. .

103

Current evidences indicate selenium as an essential trace element for

all species including humans. A positive role of selenium in human health

has been suggested. On the other hand, excess selenium is harmful and

produces toxic manifestations.

Occurrence and Distribution: Biological forms of selenium which

occur in animal body are selenium analogues of S-containing amino acids

viz. selenomethionine, selenocysteint and selcnocystine found at a mean

concentration of 02 g/g. It is widely distributed in all the tissues, highest

concentrations are found in liver, kidney and fingernails. Muscles, bones,

blood and adipose tissues show a tow concentration of selenium.

Selenium in cereal ranges from less than 0.1 g/g to 1.0 g/g wet

weight; whereas dairy products, fruits, and vegetables are relatively poor

sources of selenium. Principal source of selenium for the food is plant

material, selenium uptake in plant tissue is passive and is influenced by its

concentration in soil.

Absorption and Excretion:

Food constitutes the major route of human exposure to environmental

selenium.

Intake is in the range of 20 to 300 g/day. Infants get their selenium

through breast milk. Total body selenium has been estimated to be approx. 4

to 10 mg (average 6 mg). A good correlation between selenium intake in

food and blood levels has been shown.

104

There is also evidence to show that:

Selenium is assimilated more effectively plant food than animal

products.

Nature of diet plays a major role in determining the forms of selenium

consumed.

Other dietary constituents, e.g vit. A, C and E may also affect its

absorption.

Selenium is absorbed mainly from the duodenum and is transported

actively across the intestinal brush border particularly in the form of

methionine analogue. Selenium after absorption is transported bound to

plasma proteins particularly -lipoproteins in humans. Seleno-methionine

can be deposited directly in tissues and taken up also by myoglobin,

cytochrome C, myosin, aldolase and nucleoproteins. Selenocysteine is not

directly incorporated into proteins but is catabolized, releasing selenium for

utilization.

Main route of excretion of selenium appears to be through urine. Also

small amount is excreted through faeces and expired air.

Blood and tissue levels: Selenium levels in blood and tissues are very

much influenced by dietary selenium intake. Blood level varies 0.05 to 0.34

g/ml. Selenium levels are very low 0.05 to 0.08 g /ml in peoples of

Newzealand, where the dietary intake is approx. 20 to 30 g per day. In

selenium deficient areas of China, blood levels may be as low as 0.009

g/ml.

105

METABOLIC ROLE

1. The only metabolic role of selenium which has been established is as

the prosthetic group of selenium enzyme. Glutathione peroxidasc which is

present in cell cytosol and mitochondria and functions to reduce

hydroperoxide. |

R.OOH + 2 GSH R-OH + H2O + G - S - S – G

(Se-containing)

The reaction has special significance in the protection of

polyunsaturated F.A. located within the cell membranes where the enzyme

functions in the cytosol as part of a multi-component antioxidant defence

system within the cell. It is supplementary to vitamin E and acts as primary

antioxidant by scavenging reactive oxygen species and free radical

intermediates of polyunsaturated lipid peroxidation.

2. Selenide containing NHI proteins: Selenium probably occurs as

selenide at the active site of some non-heme iron proteins, located as integral

proteins in microsomal and other cellular membranes. Probably they are

associated with the mixed function oxidase system of membranes.

3. Relation with Vitamin E: Selenium has sparing effect on vitamin E

and it reduces the vit. E requirement at least in 3 ways:

Selenium is required for normal pancreatic function and thus the

digestion and absorption of lipids including Vit. E.

Ghutathione

peroxidase

106

As a component of glutathione peroxidase, selenium helps to destroy

peroxides and thereby reduces the peroxidation of polyunsaturated

acids of lipid membranes (discussed above). This diminished

peroxidation greatly reduces the vit. E requirement for the

maintenance of membrane integrity.

In some unknown way, selenium helps in retention of vit. E in the

blood plasma lipoproteins.

Conversely, vit. E appears to reduce the selenium supplement, at least

, in experimental animals, by prevention of selenium from the body or

maintaining it in this form. However, there are certain symptoms which be

reversed by vit. E in selenium deficient states Vit. E overcomes poor growth

of animals on the deficient diets.

4. Relation with Heavy Metals: Selenium, in comparison to sulphur,

shares an affinity with heavy metals cadmium, mercury (Hg) and silver

(Ag). Supply of selenium probably protect against toxic effects of these

heavy meals.

2.4 SODIUM/POTASSIUM PUMP

This is also called as Na+-K

+ ATPase. It requires ATP and Mg

++.

Intracellular Na+ conc. is around 10 mM/L while that of extracellular is 150

mM/L. This high inward conc. gradient is contrary to what could be

expected from the Gibbs-Donnan effect (unit -1). There is high conc. of

proteins and phosphate anions inside the cell than outside it. Conc. of K+

inside the cell is 100 mM/L while outside it is 5 mM/ L. This observation is

also unexplainable following the Gibbs Donnan effect. Na-pump is found to

107

maintain both magnitudes and direction of transmembrane concentration

gradients of those ions.

Sodium is absorbed by sodium pump situated in basal and lateral

plasma membrane of intestinal and renal cells. Na-pump actively transports

Na into extracellular fluid.

Na-pump is an enzyme Na+-K

+-ATPase. It is a glycoprotein composed

of 2 and 2 chains. Its activity depends on presence of Na+ and K

+ and

requires ATP and Mg++

ions as cofactor. The enzyme hydrolyzes a high

energy phosphate bond of ATP and uses the energy thus to transport three

Na+

ions outside and simultaneously two K+ ions inside across the cell

membrane. In this way, each Na+-K

+ pump transfers 9000 Na

+ ions outside

and 6000 K+ ions inside the cell in one minute (Fig. 2.1). The Na-pump is

very active in those cells where activities depend largely on transmembrane

Na+ fluxes, e.g. nervous, muscle fibres, renal tubules cells, intestinal

mucosal cells.

Forms of Sodium Pump and Mechanism: Na+-K

+ ATPase exists in two

forms: E1 and E2.

The E1 form: presents its ion binding and phosphate-binding sites on

the cytoplastic surface of the membrane. Three sodium ions from

cytoplasm bind with the ion binding sites of E1. This leads to the

phophorylation of aspartame residue of E1 with the help of ATP and

Mg++

. This results in conformational change and E1 becomes E2.

Now E2 exposes both ion binding and phosphate binding sites on the

extracellular surface of the membrane, lowers the affinity of the

108

ATPase for Na+ and releases it into the ECF. On the contrary, now the

K+ ions from ECF bind to the respective ion binding site of the pump.

This lowers the affinity of E2 for phosphate. This dephosphorylation

changes the conformation of E2 to El again and lowers its affinity for

K+ ions. This leads to release of the K

+ ions form ATPase into the cell.

Thus, the sodium transported actively bv Na-pump diffuses into

microvillus membrane from the lumen. Active absorption of Na+ is coupled

with glucose absorption or amino acid absorption. This carrier mediated

transport is explained in connection with Digestion and Absorption.

Fig. 2.1: Action of Na+-K

+ Pump

Excretion of Na: Every 24 hours approximately 25000 mmol of sodium are

filtered by the kidneys. However, due to tubular reabsorption less than 1% of

this sodium appears in the urine (100-200 mM/day). Approximately 70% of

the filtered sodium is reabsorbed in proximal ruble. Further 20-30% of

filtered Na+ is reabsorbed by ascending loop of Henle.

109

Check Your Progress - 1

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) Metal ions are..................to the life process. Among those

compounds are ....................., ............, numerous, ...........,

enzymes, .............. enzymes, ................ and .................. which

play important role in the metabolism of the metallic ions.

(ii) The important essential metals are: .............., ..............., .............

and ...................while important trace metals are: .................,

.............., ..................., ..................., .................., and .................

(iii)Na+/K+ pump is also called................ It maintains both

.................... and of transmembrane .................... of these ion.

2.5 TRANSPORT AND STORAGE OF OXYGEN

Multicellular organisms require large qunatities of oxygen to meet

their relatively high demands for oxidative, energy-yielding reactions.

Simple diffusion from the environment serves to bring oxygen to the cells of

plants and simple animals at a rate sufficient for their needs. However, the

cells of most animals are so far removed from the environment, and their

utilization of this material from the surface to the interior of the organism is

a necessity. In part, this mechanism involves the mechanical circulation of a

fluid, the blood, between lungs or gills and the various tissue cells. In

addition, means are usually required to increase the oxygen content of the

fluid above the low limits imposed by the poor solubility of oxygen in

aqueous media. This is accomplished by the chemical combination of

110

oxygen with a component of the circulating fluid. In most organisms this

component is an iron-porphyrin protein called hemoglobin. In the blood of

certain marine worms as iron protein complex replaces hemoglobin, while

the blood of molluscs, cephalopods, and crustacea contains hemocyanin, a

copper-protein. In all three cases the metal ion of the complex protein is the

site of oxygen-binding; however, only in the case of hemoglobin is the metal

held by a porphyrin structure.

2.5.1 Heme Proteins and Oxygen uptake

The red colouring matter of the blood is a conjugated protein.

Haemoglobin, a chromoprotein, containing heme as prosthetic group and

globin as the protein part-apoprotein. Heme-containing proteins are

characteristic of the aerobic organisms, and the altogether absent in

anaerobic forms of life.

The hemoglobins of most vertebrates have molecular weights near

68,000 and have four iron-porphyrin groups per molecular unit. Although

the protein molecules differ somewhat from organism to organism, the iron-

porphyrin prosthetic group of these proteins is a single compound, heme.

Recall that a number of other heme proteins such as catalases, peroxidases,

and cytochromes are of major biological importance. These substances are

catalysts for a variety of oxidation-reduction reactions. Only hemoglobins

and certain related compounds have the chemical property of binding

oxygen in reversible fashion. Obviously the chemical reactivity of a heme

unit (and its biological effect) is determined by the nature of the particular

protein with which it is combined and by the nature of the binding forces.

111

It is important to note that the iron atoms of the heme units of

hemoglobin remain in the ferrous state throughout the process of combining

with and releasing oxygen. These processes are not oxidation and reduction

reactions but are more properly termed oxygenation and deoxygenation. The

oxidation of the iron atoms of hemoglobin to the ferric state gives

methemoglobin, a compound lacking the property of reversible combination

with oxygen.

If you find it difficult to reconcile the structure of heme with the

usual concept of ferrous iron as a divalent ion, remember that iron, in

common with certain other metals, is capable of forming two types of bonds,

ionic and covalent. Often our attention is directed only to the ionic bonding,

so that Fe++

would be considered to have a valency of two. But a ferrous ion

can also form up to six covalent bonds; in fact, in water solution the ion is

not Fe++

but Fe(H2O)6++

. Such ions are called complex ions, and are of major

importance in biological systems in the binding of metals to proteins and

other substances. The structure of heme, in simplified form, is shown in

Figure 2.2. The N's representing the nitrogen atoms of the porphyrin ring

system. Heme is a neutral molecule in that two of the bonds of the

dispositive iron are to nitrogen atoms which can be viewed as having

negative charges.

Heme-proteins are characteristic of aerobic life. Hb is important in

O2-binding and its transport and delivery to tissues which is required for

metabolism.

Heme is a Fe-porphyrin compound. The porphyrins are complex

compounds with a "tetra-pyrrole" structure, each pyrrole ring having the

following structure;

112

HC CH

HC CH

NH

Pyrrole ring

Four such pyyroles called I to IV, are combined through

CH=bridges, called as "methyne" or "methylidene" bridges to form a

porphyrin nucleus.

Heme may be represented by following structural formula

schematically, with its attachment to globin (Fig. 2.2)

Fig. 2.2: Structure of Heme

113

The outer carbons of the four pyrrole rings, which are not linked

with the methylidene-bridges, are numbered 1 to 8. The methylidene bridges

are referred to as and,, respectively. The two hydrogen atoms in the -

NH groups of pyrrole rings (II and IV) are replaced by ferrous iron (Fe++

)

which occupy the centre of the compound ring structure and establish

linkages with all the four nitrogens of all the pyrrole rings.

The Fe, besides its linkages to four nitrogens of the pyrrole rings,

is also linked internally (5th

linkage) to the nitrogen of the imidazole ring of

histidine (His) of the polyeptide chains ("heme-linked" group). It is

considered to have a valance of six as in ferrocyanide H4Fe(CN)6 and the

sixth valence is directed outwards from the molecule and is linked to a

molecule of H2O in deoxygenated Hb. When Hb is oxygenated, the H2O is

displaced by O2.

Hb.H2O + O2 Hb.O2 + H2O

The propionic acid COOH groups of 6 and 7 positions of heme, of

III and IV pyrrole, are also linked to the basic groups of amino acids Arg

and Lys of the polypetide chains.

If the central Fe is oxidized and converted to Fe(ic) sate (Fe+++

), it

will carry a surplus + ve charge which is balanced by taking an - OH group

from the medium. It may also be balanced by other anions like Cl- or SO4

-

etc. if available (see haematin formation).

In addition, the hydrogens at positions 1 to 8 are substituted by

different groups in different compounds. In the protoporphyrin IX, which

forms parent compound of heme, the positions 1 to 8 in pyrroles are

114

substitutued by methyl (-CH3), vinyl (-CH = CH2), methyl, vinyl, methyl,

propionic acid (-CH2-CH2-COOH), propionic acid and methyl groups in that

order respectively.

2.5.2 Structure & Function of Hemoglobin

Hemoglobin, a conjugated type of protein, is the red colouring matter

of the blood. Its normal concentration is 14-16 gm./100 ml. of blood.

Hemoglobin is .found to be present in special cells, known as red blood

corpuscles (RBC). This blood protein plays an important function in the

phenomenon of respiration as it carries oxygen from the lungs to the tissues

and carbondioxide from tissues to lungs.

It is chromoprotein (type of conjugated protein), the protein part is

globin (94%) and prosthetic group is heme which is an iron (ferrous)

complex of protoporphyrin. The two moities of hemoglobin can be separated

from each other by means of dilute acid.

Heme, the coloured component of hemoglobin, is an iron (ferrous)

complex of protoporphyrin The protoporphyrin nucleus in turn is composed

of four substituted pyrrole nuclei linked by means of methine (= CH)

groups on the -positions.

Synthesis of Hb appears to proceed concurrently with the maturation

of erythrocytes. The primitive red cells contain free porphyrins rather than

Hb. As the red blood cells mature, the content of free porphyrin decreases

and that of Hb rises. These biochemical changes are correlated with the

alterations in the staining properties of the cells. Regulatory mechanisms

exist which coordinate the synthesis of heme with that of globin. Heme has

been shown to stimulate the synthesis of 'globin' on the ribosomal level. In

115

an adult human of 70 kg body wt, approx. 6.25 gm of Hb (90 mg/kg) is

synthesized and degraded per day, corresponding to approx. 300 mg of

porphyrin (porphyrin rings are about 4% by wt. of Hb molecule).

Globin

Hemoglobin Protoporphyrin

Heme

Fe (II)

Fig.2.3

Haemoglobin is a red pigment of blood. It has two part: globin and a

histidine group (Fig. 2.3). As has been mentioned above in hemoglobin,

heme nucleus remains attached via its iron with the histamine residues of the

globin molecule by means of coordinate linkage.

The giobin molecule (an example of histones) consists of four peptide

units, arranged in tetrahedral configuration; it is the nature of the four

peptides which differentiate the different types of hemoglobins (discussed

further). Most of the hemoglobins contain two identical chains and other

chains which may be and,, (epsilon). About 98% of the total adult

human hemoglobin contains two chains and two -chains. The two

members of each pair possess identical chemical composition. This type of

hemoglobin is known as hemoglobin A or A1, and is designated as

4

2

4

222 or . An chain has 141 amino acids and a molecular weight of

15,126 ; while a -chain has 146 amino acids and molecular weight of

15,866. Thus on the whole the globin molecule contains (2x 141 + 2 x l46)

574 amino acids. The complete amino acid sequence in the and chains of

hemoglobin has now been established.

116

Each polypeptide chain has one heme molecule which is suspended

between two histidine residues. One of the histidine residues (number 87 in

the -chain and 92 in the -chain) is linked directly to the iron atom of

heme, while the other histidine residue (58 in the -chain and 63 in the -

chain) is linked with the iron atom but with a gap between them into which

the oxygen molecules are introduced during the formation of

oxyhemoglobin. Thus each globin molecule containing four polypeptide

chains possess four heme molecules to form hemoglobin.

Heme Heme

Globin

Heme Heme

Thus, it is a globular protein which is composed of polypeptide

chains. These chains are arranged in a regular tetrahedral form and are

attached with four rings of pyrole (Fig. 2.2).

Functions:

(i) It is essential for O2 carriage.

(ii) It plays important part in CO2 carriage.

(iii) It is important buffering system of blood.

(iv) Various pigments of bile, stool and urine are formed from it.

In has been indicated that the outstanding property of the hemoglobins

is their reversible interaction with oxygen. This can be visualized in

simplified fashion as occurring in the following way:

HHb + O2 H+ + HbO2

-

hemoglobin oxyhemoglobin

117

The combination occurs at the unpaired electrons of iron in the heme

portion. Now since each hemoglobin molecule has four heme molecules, one

hemoglobin molecule can combine with four molecule of oxygen.

In hemoglobin at least one, and probably both, of the water molecules

of the heme structure are replaced by groups of the protein, in all likelihood

nitrogen atoms of histidine side-chains. The combination of hemoglobin

with oxygen results in the replacement of one of these groups by the oxygen

molecule. The oxygenated form is usually called oxyhemoglobin; the

deoxygenated form, simply hemoglobin. A diagrammatic version of

oxyhemoglobin appears in Figure. 2.4.

During bonding with dioxygen, the first step is the linking of O2 with

hem group :

FeII + O2 Fe

II O

O

Bonded dioxygen in the second step, coordinates with second heme,

resulting in formation of -peroxy complex:

FeII O + Fe

II Fe

II

O O

O FeII

The peroxy complex decompress to give ferryl complex of Fe(IV):

FeII

O

O + FeII 2 Fv

IV = O

118

In the end the ferryl complex combines with other heme to give

Hematin:

FeIII

= O + FeII Fe

III O

FeII

Fig. 2.4: Formation of oxyhaemoglobin

In haemoglobin, the globin part is in combination with histadine

nitrogen of protein, through Fe(II) of hem group. Thus, there are five

nitrogen in the coordination sphere of iron (four from porphyrin ring and the

119

fifth from histamine), and the sixth coordination position is available for O2

or H2O.

In heme, Fe(II) (coordination number 5) is in high spin state, which

has radii quite large to be accommodated in the ring of four nitrogen plane,

(Radius of Fe(II) is 78 pm). Hence, Fe(II) atom is forced to stay 80 pm

above the heme-group towards the adjacent histaine, like a dumb (Fig. 2.5).

Fig. 2.5

When dioxygen, O2, links as a sixth ligand Fe(II) (coordination

number 6) comes in low-spin state, which has radii 17 pm less than tha tof

high-spin Fe(II). Thus, it gets fitted in the porphyrin hole.

2.5.3 Myoglobin

The red color of many animal tissues as seen in a meat market is not

due to the hemoglobin of blood but to a related compound called myoglobin.

This compound is located within the various cells and therefore is not

involved in oxygen transport from place to place in the organism. Rather it

seems to serve as an emergency store of oxygen. Generally, those cells

which are more active in metabolic transformation have higher myoglobin

contents.

120

Myoglobin has a molecular weight of about 17,000 and contains only

one heme unit per molecule. Its protein -chain contains 153 amino acids

and the -chains are similar to that of haemoglobin only a-chains are

different. It combines more strongly with oxygen than does hemoglobin, and

releases the oxygen only when the free oxygen concentration drops to rather

low levels. The brown color of meat which has been unduly exposed to

oxygen, as in long storage or in cooking is due to the oxidation of the iron to

the ferric stage, giving metmyoglobin.

Similar to haemoglobin, its sixth coordination is also vacant; hence

dioxygen molecule can coordinate at this position reversible. At high

pressure, like haemoglobin, myglobin also binds O2 efficiently. However, at

low pressure it binds O2 at relatively faster rate. Due to utilization of

oxygen, the pressure of oxygen in muscles reduces, resulting in assimilation

of CO2 in tissues. This also reduce pH. As a result removal of oxygen from

haemoglobin and getting into myoglobin becomes easier.

In myoglobin Fe(II) remains in the high spin state, hence its radius is

approximately 92 pm and the coordination number five. Thus the

mechanism of linking with O2 is analogous to haemoglobin and the

molecular state of dioxygen in oxymyoglobin is similar to that in

oxyhaemoglobin. The Fe-O bond-lengths are equal (- 180pm) in both these

compounds, but Fe-O-O bond angle in oxymyoglobin is -115o

(while it is -

153o in human haemoglobin).

Physiology of Myoglobin and Haemoglobin:

In vertebrates when blood enters in to lungs (or gills), the partial

pressure of dioxygen there is relatively high. When it reaches in the tissues

121

of red blood cells, the partial pressure is quite less there; hence the following

reactions take place:

In lungs : Hb + 4O2 Hb (O2)4

In Tissues : Hb(O2)4 + 4Mb 4Mb(O2) + Hb

Thus, haemoglobin is ambivalent here, it strongly combines with

dioxygen and transfers it to tissues as far as possible. During this process it

is easily accepted by myoglobin, which stores it for oxidation of food. This

transfer is due to higher affinity of myoglobin for dioxygen, as compared to

that of haemoglobin. The stability constant of myoglobin-dioxygen

complixation may be given as-

][][

)]([

2

2

OMb

OMbKmb

In cells concentration of oxygen is quite low and myoglobin can

combine with it, even in low concentrations of oxygen, resulting in sufficient

quantity of oxymyoglobin. As this is not possible in case of haemoglobin, it

becomes saturated with doixygen in lungs and becomes deoxygenated in

capillaries. Haemoglobin shows dependence on pH, but myoglobin does not.

Thus, the difference in the behaviors of haemoglobin and myoglobin

towards dioxygen is related with the structure and mobility of four chains.

2.5.4 Hemoglobin and Haemerythin

Haemocyanins are iron proteins which have no heme but they

transport oxygen. In each sub unit of hamocynin, there is a pair of copper

atom. Haemocyanin, binds and transports oxygen in mollusca and

122

orthopodophyla species. These are macromolecules which have generally

more than 10037 units and molecular weights in between 25000 to 75000.

The pair of copper atoms in haemocyanins bind dioxygen molecule.

The protein is colorless when it does not has oxygen, but when it binds

oxygen it is blue, because Cu(I) is oxidized into Cu(II). Oxyhaemocyanins,

although are completely magnetic, but give antimagnetic reactions on copper

centers.

In oxyhaemocyanins the two copper atoms are separated at a distance

of 367 Ao, and are linked with three histine ligands and peroxy group and

phenolate of tyrosine (Fig. 2.6).

Fig. 2.6: Active positions in oxyhaemocynin.

The analysis of EXAS data of and compounds of Megathura

crenuleta and flelix pomatia indicates that two histidine ligands and two x

ligands (N or O) are present on each copper atom at a distance of 3.55 Ao.

Copper atoms are almost in a square planar geometry and are linked sharing

X and peroxide oxygen. The coordination number of these (two) copper

atoms is low but these are quite suitable to bind dioxygen molecules. After

oxidation of copper (I) into Cu(II), these copper atoms are interlinked

through X atoms. The peroxide linked to these copper is in binuclear state.

123

Haemerythin:

Haemerythin is used for transportation and storage of dioxygen in

different nonvertebrate species of sea. Oxygen-less haemerythin is

colourless or red and it turns pink after oxidation. The haemerythis of

Golfingia goulie insect has molecular weight approximately 108000 and is

made of eight similar subunits. Each of these sub unit has two iron atoms

linked with an oxygen molecule. Myohaemerythin, present in tissues of

muscles is a mononumeric species. Oxymyohaemerythin is more stable, just

as myoglobin is than haemoglobin.

Oxygen-less haemerythin after reaction with dioxygen oxidises the

two Fe(II) centers into Fe(III) linked with peroxide.

Oxyhaemerythin in solution and by oxidation gives Fe(III), which

does not react with oxygen, although Fe(III) links with anions. Probably

these anions bind in place of O2:

Fe(II) - Fe(II) + O2 Fe (III) - Fe(III) + O2-

Deoxy L-

Fe (III) Met Fe (III) + O2-

[Fe (II) . Fe (III)]8 (Fe (II))4 . (Fe III)4

Oxygen-less haemerythins are diamagnetic and have two high spin

Fe(II) centers. The two peroxide atoms have different atmosphere and the

Fe(III) centers different.

2.5.5 Model Synthetic Complexes of iron, Cobalt and Copper

Now a days number of model synthetic complexes of iron, cobalt and

copper have been prepared for reversible dioxygen transportation.

124

The reaction of Co(II) complexes with oxygen in solution has been

studied in detail. Under special conditions Co(II) in these complexes is

oxidised into Co(III). Intermediate perxo (O2-) and superoxo (O2

-) species of

these complexes can be obtained in absence of charcoal or other catalysts or

selecting suitable ligands.

Porphyrin complexes of cobalt also activate dioxygen. The complexes

with polydentate ligands react with DNA helix in photochemical states (Fig.

2.7).

Fig. 2.7

Binuclear Cu/Co complexes act as working model for cytochrome C

oxidase. In natural enzymes, in place of cobalt, copper atoms are linked with

histidine residue and heme iron. This involves catalytic reduction of four

electrons of oxygen in aqueous medium.

Different cobalt amines are biologically active. In human body 5 mg

cobalamine is present. Its deficiency results in different diseases. For

125

example Cobalt (III) give cobalamine with benzamidazole nitrogen ligand

and has octahedral geometry.

Simple iron (II) porphyrins oxidise readily, first into superoxo, and

then -oxodimer :

This can be achieved at lower temperature, which hinder in formation

of iron(II)-porphyrin. Under hydrophobic atmosphere reversible oxidation is

hindered. In contrast, model porphyrin must be five coordinated and most

have tendency to bind O2. For this one end of porphyrin should be restricted

to hydrophobic (lyophobic) structure.

The first example of iron (II) porphyrin, which has property of

dioxygen transportation, is a polymeric matrix of heme emidazole mixture

(Fig. 2.8).

Fig. 2.8

126

Check Your Progress - 2

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) Multicultural organisms require large quantities of oxygen to meet

their relatively.............for accomplished by................ oxygen with

a component of the..................

(ii) The red colouring matter of the blood is a...................protein,

hemoglobin, containing...................as prosthetic group and............

as the protein part.

(iii) The oxidation of the iron atoms of hemoglobin to

the...........gives................., a compound lacking the property of

..........................with oxygen.

(b) (i) When hemoglobin is oxygenated, the.....................is displaced via

by..........................

(ii) In hemoglobin.....................nucleus remains attached its............

with the....................of the...................by means of...................

(iii) Myoglobin seems to serve as an.....................of oxygen. Similar

to hemoglobin its......................is also..................., hence

dioxygen molecule can..................at this....................

(iv) Hemocyanins are ......................... which have no ............... but

they transport....................while haemerythin is used for...........

and..................of....................in different............species of sea.

127

2.6 LET US SUM UP

By going through this unit you would have achieved the objectives

stated at the start of the unit. Let us recall what we have discussed so far:

Metal compounds are closely related to the life process. Among these

compounds are haemoglobin, chlorophyll, numerous haematn

enzymes, metal activated enzymes, vitamin B12 and those vital but

poorly understood complexes which play an important role in the

metabolism of the metallic ions.

It is observed those are at least 29 different types of elements in our

body. The metals of the body are divided in two groups: (a) Essential

and (b) Trace metals.

Essential metals are those, the daily requirement of which is >

100 mg. The deficiency of these can prove fatal. These include Na, K

Ca and Mg.

Trace metals are required in less than 100 mg per day, but they are

essential, as their deficiency can lead to serious disorders. They

include Fe, Co, Cu, Cr, Mn, Zn, Se, etc.

Calcium is an important metal mainly found in bones and teeth.

Magnesium is extremely essential for life and is present as extra

cellular ion in all living cells and tissues. While sodium is the chief

electrolyte which is found in large concentrations in extra cellular

fluid compartment. Potassium is the major intracellular cation.

Trace metal ions are also called oligometals and include Fe, Cu, Co,

Zn, Mn, Me and Se.

128

Na+/K

+ pump is also called as Na

+/K

+ ATPASE. Is requires ATP and

Mg++

, Na-pump is found to maintain both magnitude and direction of

transmembrane concentration gradients of these ions.

Multicellular organisms require large quantities of oxygen to meet

their relatively high demands for oxidative, energy-yielding reactions.

In most organisms the iron-porphyrin protein called haemoglobin is

responsible for transportation of oxygen.

The red colouring matter of the blood is a conjugated protein,

haemoglobin, a chromo protein contain heme as prosthetic group and

globin as the protein part apoprotein. Haemoglobin is important in O2

– binding and its transport and delivery to tissues which is required for

metabolism.

It has been indicated that the outstanding property of the haemoglobin

is their reversible interaction with oxygen:

HHb + O2 H+ + HbO2

-

Haemoglobin Oxyhaemoglobin

In haemoglobin there are five nitrogen in the coordination sphere of

iron (four from porphyrin ring and the fifth from histine) and the sixth

coordination is available for O2.

Myoglobin is located within the various cells and therefore is not

involved in oxygen transport from place to place in the organism.

Rather it seems to serve as an emergency store of oxygen.

In vertebrates when blood enters into lungs, the partial pressure of O2

there is relatively high. When it reaches in the tissues of red blood

129

cells, the partial pressure is quite less there; hence the following

reactions takes place:

in lungs: Hb + 4O2 Hb (O2)4

in tissues: Hb(O2)4 + 4 Mb 4 Mb(O2) + Hb

Haemocyanins are iron proteins, which have no hame but they

transport oxygen. In each subunit of haemocyanin there is a pair of

copper atom. Hameocyanin bind and transport oxygen in mollusca

and orthopodophyla.

Haemerythin is used for transportation and storage of dioxygen in

different nonvertebrate species of sea.

Now a day number of model synthetic complexes of iron, cobalt and

copper have been prepared for reversible dioxygen transportation.

These include porphyrin complexes of cobalt, binuelear Cu/Co

complexes, cobalamines, iron (II) porphyrin complexes, etc.

2.7 CHECK YOUR PROGRESS: THE KEY

1(a)(i) closely related

haemoglobin

chlorophyll

haematin

metal activated

metal complexes

(ii) Ca, Mg, Na and K

Fe, Co, Cu, Zn and Mn

(iii) Na+/K

+ ATPaSe

130

magnitude

direction

concentration gradient of these ions

2(a)(i) high demands

oxidative

energy yielding

chemical combination

circulating fluid

(ii) Conjugated

heme

apoprotein

ferric state

methemoglobin

reversible combination

(iii) ferric state

methemoglobin

reversible combination

(b)(i) H2O

O2

(ii) heme

iron

histidine residue

globin molecule

coordinate linkage

(iii) emergency store

sixth coordination

vacant

131

coordinates

position reversibly

(iv) iron proteins

heme

oxygen

transportation

storage

dioxygen

non vertebrate.

132

UNIT-3 BIOENERGETICS

Structure

3.1 Introduction

3.2 Objectives

3.3 Standard Free Energy Change in Biochemical Reactions

3.4 Exergonic and Endergonic

3.5 Synthesis of ATP from ADP

3.6 Hydrolysis of ATP

3.7 Bioenergetics and ATP Cycle

3.7.1 DNA Polymerisation

3.7.2 Glucose Storage

3.8 Metal Complexes in Transmission of Energy

3.8.1 Chlorophylls

3.8.2 Photosystem-I

3.8.3 Photosystem-II

3.9 Let us sum up

3.10 Check Your Progress : They Key

133

3.1 INTRODUCTION

Energy may be defined as the capacity to perform work. Within a

system, whether this be the universe, a living cell, or a molecule, energy

cannot be created or destroyed. Energy can be transferred, however, from

one system to another in a variety of ways, such as by thermal, mechanical,

electrical, or chemical means. It is only in conjunction with the transfer of

energy by one of these means that work can be performed.

Bioenergetics or biochemical thermodynamics is the study of energy

changes in biochemical reactions. Non-biologic systems use heat energy to

accomplish work but biologic systems are isothermic and utilise chemical

energy for the living process.

Free energy (G) is the useful energy also known as the chemical

potential.

The first law of thermodynamics states that the total energy of a

system plus its surroundings remains constant. This is also the laws of

conservation of energy. Energy may be transferred from one part to another

or may be transformed into another form of energy.

The second law of thermodynamics states that "the total entropy of a

system must increase if a process is to occur spontaneously". Entropy

represents the extent of disorder of the system and becomes maximum when

it approaches true equilibrium. Under constant temperature and pressure, the

relationship between the free energy change (G) and the change in entropy

(S) is given by the following equation which combines the two laws of

thermodynamics.

134

G = H - TS

Where H = the change in enthalpy (heat) and T = the absolute

temperature.

Under biochemical reactions H is approximately equal to E. So the

above relationship may be expressed in the following manner :

G = H - TS

If G is negative is sign, the reaction proceeds spontaneously with

loss of free energy i.e. it is exergonic. On the other hand, if G is positive,

the reaction proceeds with the gain of energy i.e. it is endergonic. If the

magnitude of G is great, the system is stable. If G is zero, the system is at

equilibrium.

3.2 OBJECTIVES

The main objectives of this unit is to discuss energy changes during

biochemical reactions. After going through this unit you would be able to:

discuss standard free energy change and its importance in biochemical

reactions.

distinguish between exergonic and endergonic reactions.

describe synthesis of ATP from ADP,

discuss hydrolysis of ATP,

explain importance of ATP-Cycle in bioenergetics,

discuss part played by metal complexes in transmission of energy and

explain chlorophyll structure and its role in photosystems-I and

photosytstems-II.

135

3.3 STANDARD FREE ENERGY CHANGE :

The concept of free energy arose as a result of the search by physical

chemists for a means of predicting whether a particular reaction of series of

reactions could occur spontaneously. At first it was thought that the heat of

reaction provided the desired criterion. Generally, reactions which evolve

heat are spontaneous; those which absorb heat are not. However, exceptions

were found – certain spontaneous reactions in which the reacting molecules

absorbed heat from the solution.

The desired criterion was found only when a different kind of energy

– called entropy – was also taken into account. The total entropy content (S)

of a system is related to its relative degree of order or disorder. A system

that can exist in many possible forms is more disordered (or more random)

than is one that can exist only in a few different forms or patterns. The

higher the degree of order, the lower the entropy; the more random the

system, the higher the entropy. Thus a protein molecule will have a lower

entropy content than will the system of all if its constituent amino acids in

the free state. In considering energy changes in going from a set of amino

acids to a specific protein, it is necessary to consider both the heat of

reaction and the change in entropy.

In quantitative terms, the criterion of thermodynamic spontaneity was

found to be H - TS, where S is the entropy change, T is the absolute

temperature, and H is the heat of reaction. The TS represents that part of

the total energy of a reaction which is not available for the performance of

work (at a constant temperature). The change in available energy was then

called free energy change, and designated as G. Thus we have the equation.

136

G = H - TS

Let us consider a solution of two compounds, A and B, each of which

is present in a standard concentration of 1 mole per litre of solvent (1 molal).

Assume that these compounds can react completely to form compounds

Cand D, which will again be at unit concentrations, i.e.,

A+B C + D

Under these particular conditions of concentration, the change in free

energy is termed the standard free energy change and is indicated as Go.

If we further assume that the G0 for this process is a negative value,

we will be dealing with a conversion that could occur spontaneously.

However, this does not mean that on mixing A and B the products C and D

are formed immediately in unit concentrations. First, neither the sign nor the

magnitude of the free energy change has anything to do with the reaction

rate. For example, the Go for the complete oxidation of glucose is a very

large negative value, yet a sample of glucose may quite safely be exposed to

air without fear of an explosion. The free energy change is simply a measure

of the usable energy involved if the reaction does occur as it is written.

Secondly, the reaction will usually go only to equilibrium, not to

completion. For any process the equilibrium point is the lowest energy level.

Any reaction will proceed spontaneously to equilibrium (G is always

negative) but will be able to proceed from the equilibrium position to

completion (G is always positive) only if energy can be made available in

some suitable way. The Go for the over-all process tells us whether the free

137

energy released in going to equilibrium is greater or less than the needed to

go from equilibrium to completion.

In essence, the over-all free energy change is simply an expression (in

terms of energy) of the relative distances from the starting point to

equilibrium and from the end-point to equilibrium. To go back to our

example of the reaction A + B C + D, let's assume that going from A+B

to equilibrium involves 10 Kcal of free energy, while going from

equilibrium to C + D involves 5 Kcal. The first step is capable of supplying

all of the energy required for the second step.

In essence, the over-all free energy change is simply an expression (in

terms of energy) of the relative distances from the starting point to

equilibrium and from the end-point to equilibrium. To go back to our

example of the reaction A+BC+D, let's assume that going from A+B to

equilibrium involves 10 Kcal of free energy, while going from equilibrium

to C+D involves 5 Kcal. The first step is capable of supplying all of the

energy required for the second step,

A+B -10kCaG A+B+C+D+ 10kCaG C+D; net G=-5kCal Equilibrium mixture

plus an extra 5 Kcal. On the other hand, if we were to start with C + D, the

reaction will again proceed to the same equilibrium point, with the release of

5 Kcalof free energy. This is not enough to carry the reaction to completion,

since this would require 10 Kcal.

C+D -10kCaF A+B+C+D+ 10kCaG A+B, net G=+5 Kcal.

138

We can therefore say that the conversion of A+B to C+D, with a G

of – 5 Kcal, is a spontaneous reaction, while the conversion of C + D to A +

B, with a G of 5 Kcal is not. Be sure to note that, if we start with C + D,

appreciable formation of A and B will always occur in spontaneous fashion,

but the equilibrium will lie toward the side of C and D.

The free energy change of a reaction can be viewed simply as an

indication of where the equilibrium point lies. This should be kept in mind.

Otherwise it may be forgotten that reactions with positive G's can

nevertheless proceed to equilibrium. In fact the standard free energy change

has a very simple relation to the equilibrium constant for a reaction :

Go = - 4.6 X T X log K

In this equation T is the absolute temperature and K is the equilibrium

constant.

The application of free energy values to biological systems is fraught

with difficulties. Among other problems, the concentrations (more properly

the activities) of the reactants and products are rarely known with much

precision. These values must be known in order to correct the standard free

energy changes, which may be obtained from tables in the chemical

literature, for the exact situation being considered. It should be apparent that

the free energy change will vary with concentration of the reactants, because

the equilibrium point will change.

Secondly, the thermodynamic considerations upon which G values

are based require that the processes involved be conducted under reversible

conditions. Yet biological systems continually excrete compounds such as

139

carbon dioxide, eliminating their effect on the chemical reactions of the

system.

Furthermore, in any sequence of reactions such as characterize

metabolism, an individual reaction may have a large positive G. It is

necessary only that the G of the entire sequence have a negative value. The

step with the unfavourable equilibrium proceeds spontaneously because the

products are continually removed by succeeding reactions. For example, the

oxidation of malic acid by DPN+ has a very large positive G yet the

reaction proceeds readily in cells, primarily because the DPNH is reoxidized

by the electron transport system, a process with a large negative G.

The finding that a reaction has a positive G, therefore, does not mean

that the reaction does not occur in the living cell. Any reaction, regardless of

the free energy change, can occur provided it is followed by reactions with a

net release of free energy. These considerations apply particularly to

reactions which lead to products which are excreted from the cell.

However, processes which lead to end-products which are retained in

the cell in large quantities pose special problems. Examples of such end-

products are the polysaccharides and the proteins. The G for the

condensation of sugar units to give a polysaccharide or of amino acids to

give a protein is a very large positive value. Here there are no following

reactions to remove the products. Technically some polysaccharide and

polypeptide would be formed by equilibrium processes, but the

concentrations of glucose or of amino acids would have to be very high to

obtain even a trace of the polymeric substances. In cells, the opposite

situation occurs – the polymer content is very high compared with the

concentrations of free glucose or of amino acids.

140

In cases such as this a positive free energy change is clear evidence

that the reactions cannot occur to a significant extent by the simple

condensation of monomers to give polymers. But polymers such as proteins,

nucleic acids and polysaccharides are certainly synthesized by cells. The

answer to this apparent puzzle is that the polymers are synthesized by

mechanisms other than the condensation of monomers – mechanism which

do have an overall negative change in free energy.

3.4 EXERGONIC AND ENDERGONIC

If G for a given reaction is negative, the reaction is spontaneous and

is said to be exergonic; that is, free energy is released. For a reaction which

is not spontaneous, free energy must be supplied to the system if the reaction

is to occur. Thus G will have a positive value and the reaction is said to be

endergonic. Technically, these terms should replace those of "energy-

yielding" and "energy-requiring" reactions.

The vital processes (Synthetic reactions, muscular contraction, never

impulse conduction and active transport) obtain energy by chemical linkage

or coupling to oxidative reactions.

Metabolite A is converted to metabolite B with the release of free

energy. It is coupled to another reaction in which free energy is required to

convert metabolite C to metabolite D. Some of the energy liberated in the

derivative reaction is transferred to the synthetic reaction. The exergonic

reactions are termed Catabolism (the breakdown or oxidation of fuel

molecules), whereas the synthetic reactions are termed Anabolism.

If reaction has to go from left to right, then the overall process must

be accompanied by loss of free energy as heat. One possible mechanism of

coupling is shown below:

141

A+CIB+D

Some exergonic and endergonic reactions in biologic systems are

coupled in this way. An extension of the coupling concept is provided by

dehydrogenation reactions which are coupled to hydrogenations by an

intermediate carrier.

The alternative process of coupling from an exergonic to an

endergonic process is to synthesize a compound of high energy potential in

the exergonic reaction and to incorporate this new compound into the

endergonic reaction, thus effecting a transference of free energy from the

exergonic to the endergonic pathway.

In the living cell, the principal high energy intermediate or carrier

compound (designated-E) is ATP.

Autotrophic organisms couple their metabolism to some simple

exergonic process in their surroundigns eg. green plants utilize the energy of

sunlight and some autotrophic bacteria utilize the reaction Fe++Fe

+++.

Heterotrophic organisms obtain free energy by coupling their

metabolism to the breakdown of complex organic molecules in their

environment.

In all the three processes, ATP plays an important role in the transfer

of free energy from the exergonic to the endergonic processes. ATP is a

nucleotide consisting of adenine, ribose, and three phosphate groups. In its

reaction in the cell, it functions as the Mg++

complex.

ATP was considered to be a means of transferring phosphate radicals

in the process of phosphorylation. Lipmann introduced the concept of "high-

energy phosphates" and the "high-energy phosphate bond."

142

3.5 SYNTHESIS OF ATP FROM ADP

The energy-transfers which occur in living cells do not result in the

liberation of raw energy but involve reactions which transfer chemical

energy from one molecule to another. If the energy available from an

energy-yielding process is to be utilized to drive an energy-requiring

process, it is necessary that a single chemical compound be a product of the

energy-yielding process and a reaction in the energy-requiring process. The

ideal state is approached in living systems with the use of adenosine

triphosphate. All living cells employ this compound, commonly abbreviated

as ATP, as the major intermediate in energy-transfer processes.

Most of the ATP synthesized from ADP and P by living organisms is

derived from two major processes. One of these is based on the oxidation of

organic substances in the mitochondria of cells; the other on the utilization

by chloroplasts of radiant energy in photochemical reactions. We shall see

that these two processes are actually closely related, the significant reactions

in both cases being the phosphorylation of ADP in the course of electron

transfer from one compound to another.

143

The production of ATP from ADP and inorganic phosphate under

biological condition requires about 10 Kcal of energy per mole of ATP

formed.

ADP + Pi + 10 KCal ATP + H2O

Thus, energy is transferred to ADP and Pi to produce ATP. This

involves formation of an acid anhydride linkage. During transformation of

ATP back to ADP, an acid anhydride linkage is cleaved, and the energy is

transferred from ATP to another process, regenerating ADP and Pi

(ATP/ADP cycle).

The enzyme adenylate Kinase is present in most cells. It catalyses

interconversion of ATP and AMP on one hand and ADP on the other.d

3.6 HYDROLYSIS OF ATP

ATP is an energy rich compound. It contains two oxygen to

phosphorus bonds, called high energy phosphate bonds, represented by a

wavy line in Fig. 3.1 or P. ATP is an energy rich molecule, because the four

negatively charged Oxygen atoms in ATP are very close and the repulsive

force between them is high. Hydrolysis of a bond reduces the repulsive force

and releases a large amount of free energy:

Fig. 3.1: ATP-Molecule

144

ATP Hydrolysis ADP + Pi ( OG )

or Adenosine - P ~ P ~ P Adenosine - P ~ P + Energy

ATP Hydrolysis AMP + 2Pi ( OG )

or Adenosine - P ~ P ~ P Adenosine - P + Energy

The coupling of energy-releasing reactions to energy needing

reactions in a cell serves not only to make possible reactions which are

otherwise unfavoured but provides energy for all kinds of worked performed

by a cell. These include movement of cells, contraction of muscles, uptake

of nutrients, export of molecules across membranes etc.

3.7 BIOENERGETICS AND ATP-CYCLE

In order for a living cell to maintain its normal state, a great number

of different kinds of chemical reactions leading to the synthesis of a wide

variety of cellular constituents must continually be taking place. These

reactions are chiefly energy-requiring processes. They can occur only if

energy is made available by energy-yielding reactions. There are a number

of types of reactions which yield chemical energy in a form suitable for use

in the synthetic processes of the cell.

It would be theoretically possible to have a metabolic system in which

each one of the various energy-yielding reactions is coupled with a specific

energy-requiring processes. However, obvious advantages would be gained

by having a single, common intermediate, one compound which could be

formed by all energy-yielding processes and in turn used in all energy-

requiring processes. This ideal state is approached in living systems with the

use of ATP.

145

The energy-yielding reactions, such as occur with the absorption of

light by chlorophyll or with the oxidation of molecules such as glucose, are

linked to the productions of ATP from adenosine diphosphate (ADP) and

inorganic phosphate iron (Pi). The energy-requiring reactions, such as the

contractions of a muscle fiber or the absorption of glucose through a cell

membrane are in turn linked to the cleavage of ATP to give ADP and

ultimately phosphate ion. In essence, energy is transferred to ADP and Pi to

produce ATP, and then the energy is transferred from ATP to another

process, regenerating ADP and Pi. It is worth emphasizing that the reactions

which involve ATP are reactions in which an acid anhydride linkage is

formed, as ADP is converted to ATP, or in which an acid anhydride linkage

is cleaved, as ATP is transformed back to ADP.

The cyclical formation and cleavage of ATP in various energy-

transfer processes is illustrated diagrammatically in Figure-3.2. Several

typical energy sources are shown; a number of others would serve equally

well for the formation of ATP.

Note that in addition to the synthesis of complex compounds, which

we have previously mentioned as needing ATP, many of the physical

activities of organisms similarly require this compound as a direct source of

energy. The processes shown are common to all animals. Plants, of course,

do not have typical muscle or nervous systems. On the other hand, some

specialized organisms utilize the energy of ATP in processes which are not

shown. Prominent examples are the production of light by the firefly and the

shocking behaviour of the electric cell. Compounds such as ATP are often

described as "high-energy" or "energy-rich" compounds. However, it should

be recognized that on an equivalent basis glucose has much greater energy

146

content than is involved in the ATP-ADP interconversion. The importance

of ATP lies not so much in its energy content, though this is important, as in

the ability of this molecule, in contrast to glucose, to participate directly in

the variety of energy-transfer reactions required by the cell. This is one

major basis for the selections of ATP by living systems as their "energy-

intermediate".

ADP

+ Muscle Action

Fig. 3.2 : The Formation and Cleavage of ATP

In Energy-transfer Processes.

Also it may be well to mention that ATP is often said to have a "high-

energy phosphate bond." The term is a very poor one but is in wide use to

indicate that the pyrophosphate (phosphate anhydride) linkage is very

reactive portion of the molecule. A better expression is the statement that

ATP has "high phosphate-group transfer potential," to indicate that the

phosphate group readily can be transferred by chemical reactions to other

compounds.

The role of ATP in biological systems was discovered in 1929 by

Lohmann. We have previously mentioned the use of iodoacetic acid as an

inhibitor in yeast of one of the reactions of glucose oxidation. This same

compound also was found to cause the rapid loss of ability of an isolated

muscle fiber of an animal to undergo contraction. Lohmann found that the

addition of ATP to the medium containing the muscle fiber restored

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temporarily the contractile ability. The way was then clear to suggest that

ATP was formed by the oxidation of glucose and utilized in muscle

contraction. This led to much experimentation concerning the role of ATP in

other systems and the detailed mechanisms for the production and use of the

compound.

Lipmann introduced the symbol – P , indicating high-energy

phosphate bond. The term group transfer potential is preferred to "high-

energy bond". Thus, ATP contains 2 high-energy phosphate groups and

ADP contains one. The phosphate bond in AMP is of the low energy type,

since it is a normal ester link:

ATP = Adenosine - P ~ P ~ P

ADP = Adenosine - P ~ P

AMP = Adenosine - P

Thus -

1. ATP is the donor or high-energy phosphate and ADP can accept high-

energy phosphate to from ATP. ATP/ADP cycle connects these

processes which generate - P to those processes that utilize – P .

2. There are three major sources of - P taking part in energy conservation

or energy capture.

(a) Oxidative phosphorylation: This is the greatest quantitative source of -

P aerobic organisms. The free energy comes from respiratory chain

oxidation within mitochondria.

(b) Glycolysis : A net formation of 2 - P results from the formation of

lactate from one molecules of glucose, generated in two reactions

catalyzed by phosphoglycerate kinase and pyruvate kinase.

(c) Cltric acid cycle : One - P is generated directly in the cycle at the

succinyl thiokinase step.

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3. Another group of compounds (Phosphagens) act as storage forms of

high-energy phosphate. These include creatine phosphate in vertebrate

muscle and brain, arginine phosphate in invertebrate muscle.

4. In physiologic conditions, phosphagens permit ATP concentrations to

be maintained in muscle when ATP is being rapidly used as a source

of energy for muscular contraction. When ATP is abundant, its

concentration can cause the reverse reaction to take place and allow

the concentration of creatine phosphate to increase abundantly so as to

act as a store of high-energy phosphate. When ATP acts as a

phosphate donor to from those compounds of lower free energy of

hydrolysis, the phosphate group is invariably converted to one of low

energy eg.

Glycerol + Adensine - P ~ P ~ P

Glycerol kinase

Glycerol – P + Adenosine - P ~ P

3.7.1 DNA – Polymerisation :

The mechanism of biosynthesis of DNA has been largely clarified by

the discovery of the enzyme DNA polymerase (or DNA nucleotidyl

transferase). This enzyme catalyses the polymerisation of the four

deoxyribonucleoside triphosphates (i.e. mononucleotides) in the presence of

Mg++

ions and a primer DNA (i.e. some natural DNA which initiates the

polymerisation). The four deoxyribonucleoside triphosphates (i.e.

mononucleotides) which act as substrate are the 5'-triphosphates of

deoxyadenosine (dATP), deoxyguanosine (dGTP), deoxycytidine (dCTP)

149

and deoxythymidine (dTTP). The overall equation of the polymerisation is

given below :

n.dATP dAMP

+ DNA

n.d.CTP + DNA Polymerase dCMP

+ + 4n (PPi)

n.dGTP dGMP

+

n.dTTP dTMP-n

DNA n

The energy required during the reaction for formation of the 3', 5'-

phosphodiester bonds of the polydeoxyribonucleotide, is provided by the

high energy bonds in the linear triphosphate units of the dATP, dCTP, dGTP

and dTTP, as each monomer is incorporated into the new chain, it loses its

terminal pyrophosphate unit (PPi).

The mechanism of DNA synthesis is illustrated in Fig. 3.3. Under the

influence of DNA polymerase, in the presence of Mg++

, the double strands

of the DNA acting as a template (or primer) separate (a small portion at a

time), by cleaving the hydrogen bonds between complementary bases. The

four deoxyribonucleoside triphosphates are attracted from solution in the

cellular sap to form hydrogen bonds with their complementary bases on the

separated strands of the primer DNA. Thus the DNA template (or primer)

dictates the sequence in which the monomers are assembled. During this

reaction each nucleotide loses a pyrophosphate group and forms an ester

linkage with the 3'-hydroxyl group of the deoxyribose on adjacent new

nucleotide via its remaining phosphate group (on the C5'). Thus two daughter

double helices are formed, each consisting of an old strand of the primer

150

DNA and complementary new strand. The final composition and nucleotide

sequence of each strand is identical with the corresponding strand in the

primer (parent) DNA. This process has been named as replication. It is

important to note that the acridine drugs (e.g. proflavine) and antibiotics of

the mitomycin type inhibit DNA replication.

Recent studies on the enzyme DNA polymerase have shown that it

has the power to repair broken DNA chains by removing in a stepwise

manner, the broken chain, starting at the 5'-phosphate terminus and re-

synthesizing a new chain. Deoxynculeotide units are added to the 3'-

hydroxyl terminus of the break using the intact chain as template.

Fig. 3.3: Mechanism of Polymerisation of DNA.

151

The Watoon-Crick double helix model of DNA-molecule is shown in

Fig. 3.4.

Fig. 3.4: Watson-Crick model of DNA-Molecule.

3.7.2 Glucose Storage

The utilization of the chemical energy of the glucose molecule for the

production of ATP will be examined as an example of the relationship

between biological oxidations and ATP synthesis. Most of the material

discussed will be applicable in principle to the mechanisms for the oxidation

of other compounds in biological systems.

Let us consider first the quantitative aspects of energy transfer during

the complete oxidation of glucose. When a mole of glucose is burned in air,

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686 kilocalories (Kcal) of energy appear as heat, light, and pressure-volume

work. The equation for the oxidation of glucose under these conditions is:

glucose + 6O2 6CO2 + 6H2O + 686 Kcal

The production of ATP from ADP and inorganic phosphate under

biological conditions requires about 10 Kcal of energy per mole of ATP

formed.

ADP + Pi + 10 Kcal ATP + H2O

Obviously, the oxidation of 1 mole of glucose can supply energy

sufficient for the formation of about 68 moles of ATP. In the oxidation of

glucose to carbon dioxide (CO2) and water by the most common metabolic

pathway, about 38 moles of ATP are actually produced by cells per mole of

glucose oxidized. The biological oxidation of glucose might therefore be

summarized as:

glucose+6O2 + 38 ADP + 38 Pi

6 CO2 + 6H2O+38 ATP + heat (about 310 Kcal)

You should realize that this equation cannot possibly represent a

single chemical reaction, but must be a summary of many reactions. As

written, the equation implies that 83 molecules collide simultaneously and

are converted in one step to the products. In actuality, very few reactions

exist which involve the simultaneously participation of more than three or

four molecules. Therefore, the production of ATP in conjunction with the

oxidation of glucose must occur in a stepwise fashion.

This means that only one molecule of ATP can be synthesized in any

single reaction, ADP being one reactant, and an inorganic phosphate ion

(indirectly) being a second. If the biological oxidation of glucose could take

153

place in one step, we would anticipate the production of only one molecule

of ATP, the rest of the energy being lost as heat. Because 38 molecules of

ATP are actually produced upon the oxidation of one molecule of glucose,

the conclusion must unavoidably be that the oxidation does not occur is one

step, but in many steps. In the step wise process, the energy of the glucose

molecule can be released in a number of ATP molecules can be synthesized.

Theoretically one ATP could be made in each step which liberates at least 10

Kcal of energy.

The sequential reactions necessary for the biological oxidation of

glucose with the concurrent production of ATP make up a major portion of

the metabolic activities of living cells. It is necessary to note that among the

sequential reactions needed for the complete oxidation of the carbon atoms

of glucose to carbon dioxide, only six oxidation reactions actually occur.

Again we are faced with a problem: How is it possible to produce 38

molecules of ATP using only six oxidation reactions?

In order to answer this questions let us consider one example of the

six oxidations, the oxidation of malic acid to oxalacetic acid. These

compounds are two of the intermediates of the metabolic oxidation of

glucose. First we shall give the reactions as if it were a single step:

154

The reaction pictured is the minimum degree of oxidation of an

organic compound which gives a stable product. Two electrons and two

hydrogen atoms have been removed and accepted by the oxygen atoms (in

O2 the oxidation state or net valence is zero, in H2O the oxygen has an

oxidation state of minus 2). Single electron changes would give unstable free

radicals as products.

Note that even though the malic acid has been subjected to the

minimum possible oxidations, the energy released is sufficient to form four

molecules of ATP. As indicated above, we could actually expect only one

ATP to be formed if the reaction occurred in the single step pictured. Again

this would be wasteful of energy because most of it would be lost as

unavailable heat. Biological systems are considerably more efficient; they

form three molecules of ATP per molecule of malic acid which is oxidized.

This implies that a sequence of step wise reactions occurs, three of which are

coupled with the synthesis of ATP. The oxidation of malic acid to oxalacetic

acid, a two-electron change, is the minimum possible. Rather than passing

the electrons directly to oxygen, they are passed through a series of

oxidation-reduction reactions and only in the last step to oxygen. In the

course of this stepwise transfer of electrons, three of the steps are coupled to

ATP formation, thus making use of most of the energy of the oxidation.

Check Your Progress - 1

Notes: (i) Write your answers in the space given below.

(ii) Compare your answers with those given at the end of the unit.

(a) (i) Bioenergetics or..............................is the study of.....................in

biochemical reactions.

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(ii) Free energy................., is the useful energy also known as

the................ If Gis negative in sign, the reaction.................. with

loss of free energy i.e. it is................ On the other hand, if G is

positive, the reaction proceeds with the..................i.e.it is..............

(iii) The energy is transferred to ADP and Pi to produce..............; while

during transformation of...............back in to.................., the energy

is transferred from...................to another process,

regenerating.................

(b) (i) ATP is an................., because the four..............atoms in........... are

very close and.............between them is.................

(ii) The mechanism of polymerisation of DNA has been largely

clarified by the discovery of the enzyme...................which

catalyses, the............of the four....................in the presence

of................ions and a..............

(iii) The oxidation of glucose to....................and..................can supply

energy sufficient for formation of about.

3.8 METAL COMPLEXES IN TRANSMISSION OF ENERGY

Metal complexes (Coordination compounds) are closely related to life

process; however the proper understanding of the coordination aspects of

biochemical processes is limited due to the complex structures of the

biochemical molecules. Eichorn underlined the participation of coordination

compounds in almost every phase of biological activity. All these processes

involve one or more enzymes, which need metallic ions for their activity and

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have been shown to function as metal complexes e.g. in the natural process

of bond formation and bond rupture (cleavage of peptide bonds or

cargboxylation and decarboxylation reaction, or phophorylation reaction i.e.

conversion of ATP to ADP etc); the exchange or blocking of functional

groups; in influencing sterochemical configuration; in oxidation-reduction

reaction; in storage and transfer reactions (transportation of oxygen i.e.

functioning of haemoglobin) and in transmission of energy.

For transmission of energy functioning of chlorophyll (Mg2+

complex)

during photosynthesis is an important example.

3.8.1 Chlorophyll

Chlorophyll is the best known and highly special coordination

compound of plants, which takes photons from sunlight and pass it to the

system responsible for conversion into necessary energy for the chemical

change. Like haemoglobin, chlorophyll is also a metal-porphyrin complex,

in which Mg(II) ion remains coordinated with the four nitrogen atoms of

porphyrinring stated in a square planer geometry (Fig. 3.5). Chlorophyll is a

green pigment of plants, which plays important part in photosynthesis

process.

Fig. 3.5: Chlorophyll

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A double bond of a pyrrole ring in chlorophyll is reduced and the long

alkyl chain at the base of its structure is a phytyl group.

A reason for usefulness of chlorophyll in photosynthesis process is

high conjugation of porphyrin ring. This reduces the energy of electronic

transfers and shifts the absorption centre in 350-700 nm region, where one or

more photosynthetic pigments absorb light at every frequency.

The main point of the reaction centre is a pair of chlorophyll

molecules, called special pair. In the special pair, one pyrrole ring of each

molecule remains in contact with the other by overlapping. In addition to

this on each molecule an acetyl group remains coordinated with magnesium

atom of other molecule. On the sixth coordination site of each magnesium

atom, nitrogen of histidine part of the protein chain is attached. Special pair

also remains attached with pheophytin and quinone molecule, which accept

electron from the reaction centre. Near the quinone molecule is present one

non-home iron atom, which remains coordinated with four histadine and one

ghetamic acid. Electron reach in to the reduction chain through the iron

atom. The hole (the centre where electron is absent) of the reaction centre is

filled with the electron obtained from cytochrome. The separation of charge

between positive charge on Fe(III)-cyto chrome and the electron coming in

z-scheme chain represents the potential energy used in photosynthesis -

process.

Photosynthesis

Photosynthesis converts radiant energy into useful, high quality

chemical energy in the bonds that holds together organic molecules. Energy

captured by photosynthesis supports nearly all life on earth. During

158

photosynthesis, solar energy is converted into chemical energy, when

atmospheric CO2 is assimilated during the synthesis of organic molecules.

During this process, water provides the reducing power and the pigments

capture the light energy. The present status of our knowledge about

photosynthesis has been achieved through the use of techniques from a wide

variety of disciplines including physics, bioenergetics, biophysics electron

microscopy, bioenergetics, biophysical electron microscopy, biochemistry

and chemistry, plant physiology, agronomy, genetics and molecular biology.

In recent years, spectacular success has particularly been achieved in

resolving the structure of several membrane proteins associated with the

photosynthetic apparatus, so that this apparatus (mainly the type-II reaction

centre in bacteria) is the first complex biological system, for which structure,

function and regulation has already been described in rigorous

physicochemical terms at the atomic level. Therefore, now it is a challenge

for scientific community to devise low cost artificial power generating

systems capable of exploiting solar energy based on the principles of

photosynthesis. The equation of photosynthesis can be written as follows:

6CO2 + 12 H2O light C6H12O6 + 6O2 + 6H2O

Although initially it was believed that the O2 evolved during

photosynthesis is derived from CO2 later it was established beyond any

doubt that this O2 is actually derived from water. The reactions involved in

the release of O2 from water is described as Hill reactions after the name of

Robert Hill. He was the first to demonstrate the origin of O2 from H2O

(rather than from CO2) during photosynthesis according to the following

equation:

2H2O light O2 + 4H+ + 4e

-

159

The biochemical process, photosynthesis may be divided into two

main groups. The first of these involves those chemical reactions necessary

for the conversion of the radiant energy of the sun into the chemical energy

of compounds which plant cells are able to use as energy sources for the

synthesis of organic compounds. These reactions are appropriately known as

the light reactions.

The second division of the field of photosynthesis includes the

reactions by which the chemical energy of each of the compounds formed in

the light reactions is utilized to promote the formation of organic substances

from inorganic materials. These processes are termed the dark reactions.

(i) Light reaction (electron transport and generation of NADPH and

ATP) for this reaction, light is necessary. In higher plants, it takes place in

grana and stoma lamellae of the thylacoid membrane of the chloroplast. In

photosynthetic bacteria having no chloroplasts, this reaction take place in the

cell membrane containing the pigment system. This involves oxidation of

water, leading to the transfer of electrons to NADP+ and to the ultimate

production of NADPH and ATP. This light reaction takes place in protein-

pigment complexes described as reaction centres. In photosynthetic bacteria,

there is only one reaction centre, but in organisms including cyanobacteria,

algae and higher plants there are two reaction centres.

(ii) Dark reaction (carbon dioxide reduction in C3, C4 and CAM plants).

In this reaction, light is not necessary, but it can take place in the light as

well. This reactions takes place in the stroma and makes use of NADPH as

the reducing power and ATP as the source of energy for reduction of

atmospheric CO2 into carbohydrates. The CO2 assimilation can take place

160

through the synthesis of a stable compound, which is a 3 carbon compound

(phosphoglyceric acid) in majority of plants but can also be a 4 carbon

compound (oxaloacetate, malate or aspartate) as in several grasses and

Crasulacean plants (succulents). It will be shown later that the C4 pathway,

involving the synthesis of a 4 carbon compound (malate or aspirate) as

Calvin-Beason cycle, which is universally accepted. Similarly C4 – pathway

was worked out by M.D. the first stable compound, is more efficient, since it

does not allow photorespiration, which is a wasteful process for C3-pathway.

Melvin Calvin and his co-workers at the University of California worked out

the mechanism of the reduction of CO2 and suggested a cyclic pathway, now

described as Hatch and C.R. Slack in 1970 and is described as Hatch and

Slack Pathway. Metabolic pathway for CO2 fixation in CAM (Gassulacean

Acid Metabolism) plants, through resembles that in C4 plants, differs from it

in several essential features.

The photosynthetic reactions occur in tiny, membranous organelles,

the chloroplasts of plant cells. Like mitochondria, these relatively large

particulate components of the cytoplasm have complex internal structures.

Chlorophyll molecules are concentrated within the chloroplasts, in small

bodies known as grana. Each of these appears to consist of an intricately

folded lipoprotein membrane, resulting in a many-layered structure. The

chlorophyll molecules are sandwiched between layers of the lipoprotein.

Probably the long hydrocarbon chain of the chlorophyll molecule is

associated with the lilied portion of the membrane protein, while the

remainder of the molecule is linked with a water-soluble portion of the

protein. Relatively large amounts of carotenoid compounds such as -

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carotene and Vitamin K also occur in the grana, perhaps as part of the

lipoprotein membranes.

Mechanism of Photosynthesis: (Light-induced electron transport and

generation of NADPH and ATP) we earlier described that light energy is

captured during the 'light reaction' and that CO2 is reduced during the 'dark

reaction' of photosynthesis. The reduction of CO2 leading to production of

carbohydrates is the ultimate goal of photosynthesis. However, for achieving

this goal, following two co-factors are needed (i) NADPH, which, in the

form of a high-energy compound, works as an energy source and (ii) ATP,

which in the form of high energy compound. These two co-factors (NADPH

and ATP) are produced during light-induced electron transport, taking place,

in the thylakoid membrane of the chloroplasts in algae and higher plants.

The process of energy conversion in photoynthesis begins, when light

is absorbed by chlorophyll molecule locked in the light harvesting

complex. The chlorophyll molecule gets excited by a quantum of light

(photon); this involves movement of an electron from a molecular orbital of

lower energy level to another orbital of higher energy level. Such an

excited chlorophyll molecule is unstable and will tend to return to its

original unexcited stale in one of the following three ways :

(i) extra energy is converted either exclusively to heat energy (used in

movement of molecules) or to a combination of heat and light of long wave

length (used in fluorescence); fluorescence can be seen when light is

absorbed by isolated chlorophyll molecules in solution; (ii) extra energy (not

the electron itself) is transferred to a neighbouring chlorophyll molecule by a

process called resonance energy transfer and is used for increasing the

162

energy level of an electron in this neighbouring molecule; (iii) the excited

chlorophyll molecule takes the role of electron donor and transfers the high

energy electron to a nearby molecule (not necessarily a chlorophyll). This

nearby molecule performs the role of an electron acceptor. The chlorophyll

molecule, excited due to absorption of a photon, after transfer of a high

energy electron, now accepts a low energy electron from some other

molecule (electron donor) and returns to its ground stale. The last two of

these three mechanisms are utilized during photosynthesis. The first of these

last two mechanisms (resonance energy transfer) is utilized for transfer of

energy from one chlorophyll molecule to another in the light harvesting

complex (also called antenna complex), without the transfer of electron and

the second is utilized in the transfer of a high energy electron from excited

singlet state: (PD) of chlorophyll in 'special pair' to the primary electron

acceptor (PA).

In a simplified version, we may say during photosynthesis, after

decomposition of water into component elements, carbondioxide is reduced:

2H2O 4[H] + O2

x CO2 + x/2 [4H] [CH2O]x + x/2 O2

In green plants there are two paired photosynthesis systems A and B.

Both differ in the type of chlorophyll present and in the chemical using the

absorbed photon energy (the ratio of chlorophyll A and B is 3:1).

Chlorophylls absorb lower energy light (~700 nm) in far i.r. region.

When photon colloids with chlorophyll of any system (A or B) it provides

energy for the series of redox reactions.

1. Ist Step : Chlorophyll excitation :

Chl hV

Chl*

Chlorophyll

163

2. IInd

Step : Within picoseconds energy transfers in to electrophile

Chl* Chl

+ + A

-

3. Next Step : Excited acceptor transfers energy to other acceptor and

this follows :

A- + B

B

- + A

B- + C

C

- + B

After many steps:

6CO2 + 6 H2O C6H12O6 + 6O2

These redox reactions take place in a series (Fig. 3.6)

Figure 3.6 : A Summary of the Light Reactions of Photosynthesis.

164

Photosystems I and II

In higher plants and in cyanobacteria photochemical reaction centre

and the light harvesting complex(es) (also called antenna complex)

associated with it together form a photosystem. There are two such

photosystems, photosystem II (PSII) and photosystem I (PSI) embeded in

the thylacoid membrane and linked with each other through a

thermochemical bridge consisting of several electron carriers, particularly

the mobile plastoquinone (PQ) and plastocyanin (PC). The present status of

our knowledge about the structure and organization of these components

both in higher plants (also in algac and cyanobacteria) and in photosynthetic

bacteria will be briefly presented here.

Photosynthetic organisms have two tasks in early steps of

photosynthesis : (i) to capture photons with high efficiency and at a

reasonable metabolic cost and transfer them to a reaction centre and (ii) to

convert solar energy into chemical energy.

The photosystem II (PSI1) and photosystem I (PSI), mentioned

above, are multiprotein complexes. They respectively contain, type-II and

type-I reaction centres (also called trapping centres) along with their light

harvesting complexes (LHCII and LHCI). These reaction centres are

comparable to the corresponding reaction centres of photosynlhetic bacteria,

particularly for the purpose of relating them with their evolutionary origin.

These two reaction centres, in terms of their chemical constitution, are

'pigment-protein complexes' and are distinguished from each other due to

their different terminal electron acceptors. Type-I reaction centre, found in

green sulphur bacteria and heliobacteria, are characterized by having low

165

potential iron-sulphur centres as terminal electron acceptors. Type-II

reaction centre, found in anaerobic sulphur and non-sulphur purple bacteria

(e.g. Rhodopseudomonas viridis and Rhodobactsr sphaeroides) and also in

some green bacteria (e.g. Chloroflexus), on the other hand, are

characterized by having quinines as their terminal electron acceptors. As

we know, in higher plants the two reaction centres arc coupled together in

series, through a thermochemical bridge (which does not need light) so as

to oxidize water and create a low redox potential needed to reduce NADP+.

Reduced NADP+ (NADPH), with ATP arc then used to convert CO2 to

organic molecules (this involves addition of CO2 to RuBP, a 5-carbon

compound, to produce 6-carbon compound like glucose). In higher plants,

besides PSII and PSI, there are following two other membrane complexes

involved in the production of NADPH and ATP : (i) cytochromc b6f,

which acts as a redox link between PSII and PSI and (ii) coupling factors

(CF0 and CF1), which arc components of ATP synthase complex and are

involved in the production of ATP from ADP, through the use of

transmembrane proton gradient generated by light driven electron transfer

from PSII to PSI (in accordance with Milchcll's, 'chemiosmotic theory').

Both the above functions are, however, done by the pigment systems,

present in the plasma membranes in bacteria and in the thylacoid membrane

of the chloroplasts in algae and higher plants. In chloroplasts of higher

plants, there are about 60 different proteins (only half of them coded by

cpDNA), associated with the energy converting systems, but only about a

dozen of them are directly involved in the energy transduction pathways.

The pigment molecules responsible for collection of energy (ultimately for

extracting electrons from a substrate, to be transferred to an electron

166

acceptor) are a part of the light harvesting complexes designated as LH1 and

LH2 for bacteria and LHCI and LHCII for higher plants.

3.8.2 Photosystem-I : (Type-I reaction centres) The type-I reaction centre

resembles type-II reaction centre in many ways and is found in isolation in

some an-oxygenic organisms including green-sulphur bacteria and

heliobacteria. In association with type-II reaction centre (found in PSII), this

reaction centre (type-I RC found in PSI) is also found in oxygenic

photosynthetic organisms including cyanobacieria, algae and higher plants,

where aerobic photosynthesis is carried out following the Z-scheme. Like

type-II reaction centre, in type-1 reaction centre also, a 'special pair' of

chlorophylls acts as the primary electron donor. The oxidizing potential

generated in type-I reaction centre is the same (about +0.4V) in oxygenic

and anoxygenic organism, in contrast to type-Il reaction centre, where it

differs in oxygentic (+1.1V) and anoxygcnic organisms (+ 0.4V).

The type-I reaction centre is also a protein-pigment complex and has a

two-fold symmetry like type-II reaction centre, although its structure at the

atomic level has not been resolved, to the same level as that of type-II

reaction centres. However, in type-I reaction centre also, there are two

similar or identical proteins, which form dimmer. It differs from the protein

dimmer of type-II reaction centre, in having a large number of light-

harvesting chlorophylls. The low-potential (0.7V) of 4 Fe-4S (terminal

electron acceptor) reduces NAD+ or NADP

+ through other iron-sulphur

centres, which are located in proteins associated with the outer surface of

the reaction centre dimer.

167

In oxygenic photosynthetic organisms (cyanobacteria, algae and

higher plants), PSI consists of a number of intrinsic polypeptides and

extrinsic polypeptides, which include ferredoxin containing Fe2S2 centers

and ferredoxin-NADP reductase (flavoprotcin). The most important of these

is 68kD protein, which is a tetramer and contains P-700. It binds about 13

'chlorophyll a' and 6 caratenoid molecules. The AO and A1 acceptors arc also

present on this polypeptidc. Associated with four polypeptides in PSI

complex in chloroplasts, there is also a light harvesting complex I(LHCI),

whose polypeptides range from 22 kD to 25 kD (Table 13.1).

3.8.3 Photosystem-II : (Type-II reaction centres). The type-II reaction

centre is exemplified by the reaction centre found in photosynthetic purple

bacteria, Rhodopseudomonas viridis and Rhodobacter sphaeroides. In these

bacteria, the three dimensional structure of type-II reaction centre has been

studied in great detail, leading to the award of 1988 Nobel Prize in

Chemistry to J. Deisenhofer, R. Huber and H. Michel of Max Planck

Institute in Martinsried, Germany.

In higher plants PSII complex of (i) water-splitting or oxygen

evolving complex (OEC), (ii) PSII core complex (reaction centre) and (iii)

LHCUII. Four proteins of oxygen evolving system, 12 polypeptides

belonging to the core complex and five proteins belonging to the LHCII are

known. While proteins for oxygen evolving complex and LHCII are encoded

in the nucleus, those for PSIl core complex are encoded in the chloroplast

genome. Many of these proteins are integral membrane-spanning

polypeptides, but some of them are extrinsic polypeptides. The extrinsic

polypeptides can be removed by relatively simple procedures (washing with

salt solutions under appropriate pH conditions).

168

Thus in photosystem-I (Photosynthesis system-A in Fig. 3.7) the

common strong reducing specie, REDA and the strong oxidising species,

OXA are formed, while in photosystem-II (Photosynthesis system- B in fig.

3.7)

Photosynthesis System B Phtosynthesis System A

Fig. 3.7: Electron movement in photosynthesis system A and B

a strong oxidising agent, OXB but a weak reducing agent, REDB are

obtained. The function of OXB is to produce molecular oxygen. A

manganese complex, reduce OXB, which is regenerated, by an another

excited chlorophyll molecule, for further use. In this redox reaction

manganese is converted into +2, +3 and +4 states.

REDA gives its electron to a carrier like ferredoxin [Fe2+

/Fe3+

], as a

result NADPH is formed which is a stable source for reduction of

carbondioxide into carbohydrate.

In photosystem-II (Photochemical system B) the carrier is pheophytin.

The ion pair formed in step 1st (given above) are Chl

+ and Pheo

-. Here two

points are important:

169

1. The excessive conjugation of porphyry system, which is responsible

for the shift of absorption in the visible reason, also increases the strength of

the ring and saves the loss of energy due to molecular vibrations, and

2. The phosphorescent behaviour of chlorophyll for phosphorescence

presence of a metal ion is necessary. Free porphyrin gives only fluorescent

emission. Metal ion helps in mixing of excited singlet and trilpet states, thus

promotes formatron of relatively stable triplet state, which is the source of

energy for phophorescence and photosynthesis.

CHECK YOUR PROGRESS - 2

Notes: (i) Write your answers in the space given below.

(ii) Compare your answers with those given at the end of the unit.

(a) (i) Metal complexes are closely related to............... For transmission

of energy functioning of............................ during.........is an

important example.

(ii) Chlorophyll is a metal..............complex, in which............. .remains

coordinated with................atoms of................situated in a

.................. geometry.

(iii) The high ...............of..............ring in chlorophyll shifts the

absorption centre in.............region, where one or more.........

.absorb light.

(b) (i) During photosynthesis..............is converted into................, when

atmospheric...............is assimilated during the synthesis

of....................

170

(ii) The biochemical process of photosynthesis may be divided into

two groups: (a) ....................and (b)......................

(iii) There are two photosystems, photosystem-I and photosystem-II.

These have two tasks:

(a) ..............................................................

(b) ..............................................................

3.9 LET US SUM UP

By going through this unit you would have achieved the objectives

stated at the start of the unit. Let us revise what we have discussed so for :

Bioenergetics or biochemical thermodynamics is the study of energy

changes in biochemical reactions. Free energy, G, is the useful

energy also known as the chemical potential.

Under constant temperature and pressure, the free energy is related

with the enthalpy change, H and the change in entropy, S,

according to the following thermodynamic relation:

G = H - TS, where T is the absolute temperature, while under

biochemical condition H is approximately equal to E (Heat change) and

S is the measure of change in disorder of the system.

If G is negative, the reaction processes spontaneously with loss of

energy i.e. it is exergonic. On the other hand, if G is positive, the

reaction proceeds with the gain of energy i.e. it is endergonic.

Technically exergonic and endergonic terms should replace those of

energy yielding and energy requiring reactions.

171

In the living cells, the principal high energy intermediate or carrier

compound is ATP (Adenosine triphophate).

In different processes, ATP plays an important role in the transfer of

free energy from the exergonic to the endergonic processes. ATP is a

nucleotide consisting of adenine, ribose and three phosphate groups.

In its reaction in the cell, it functions as the magnesium (Mg++

)

complex.

All the living cells employ ATP as the major intermediate in energy-

transfer processes. Most of the ATP synthesized from ADP and P by

living organisms is derived from two major processes. One of these is

based on the oxidation of organic substances in the mitochondria of

cells; the other on the utilization by chloroplasts of radiant energy in

photo-chemical reactions.

Energy is transferred to ADP and Pi to produce ATP. This involves

formation of an acid-anhydride linkage. During transformation of

ATP back to ADP, an acid anhydride linkage is cleaved and the

energy is transferred from ATP to another process, regenerating ADP

and Pi (ATP/ADP- cycle).

ATP is an energy rich compound. It contains two oxygen to

phosphorous bonds called high energy phosphate bonds. The four

negatively charged oxygen atoms in ATP are very close and the

repulsive force between them is high. Hydrolysis of a bond reduces

the repulsive force and releases a large amount of free energy.

172

The mechanism of biosynthesis of DNA has been largely clarified by

the discovery of the enzymes DNA polymerase. This enzyme

catalyses the polymerisation of the four deoxyribonucleoside

triphosphates in the presence of Mg++

ions and a primer DNA. During

this the double strands of the DNA acting as a template (or primer)

separate, by cleaving the hydrogen bonds between complementary

bases.

The four deoxyribonucleotide triphosphate areattroeted from solution

in the cellular sap to form hydrogen bonds with their complementary

bases on the separated strands of the primer DNA. Thus the DNA

template (or primer) dictates the sequence in which the monomers are

assembled.

During this reaction each nucleotide loses a pyrophosphate group and

form an ester linkage with the 3'-hydroxyl group of the deoxyribose

on adjacent new nucleotide via its remaining phosphate group (on the

C5). Thus two daughter double helices are formed, each consisting of

an old strand of the primer DNA and a complementary new strand.

The double helix model of DNA molecule was proposed by Waston

and Crick.

The utilisation of the chemical energy of the glucose molecule for the

production of ATP may be examined. When a molecule of glucose is

burned in air, 686 Kcal of energy appear as heat, light, and pressure

volume work. While the production of ATP from ADP and inorganic

phosphate under biological conditions requires about 10Kcal energy

per mole of ATP formed. Obviously, the oxidation of 1 mole of

173

glucose can supply energy sufficient for production of about 68 moles

of ATP.

Metal complexes are closely related to life processes. For transmission

of energy functioning of chlorophyll, a Mg++

-porphyrin complex,

during photosynthesis is an important example.

In chlorophyll molecule Mg(II) ion remains coordinated with the four

nitrogen atom of porphyrin ring situated in a square planer geometry.

A reason for usefulness of chlorophyll in photosynthesis process is

high conjugation of porphyrin ring. This reduces the energy of

electronic transfers and shifts the absorption centre 350-700 nm

region, where one or more photosynthetic pigments absorb light at

every frequency.

During photosynthesis, solar energy is converted into chemical

energy, when atmospheric CO2 is assimilated during the synthesis of

organic molecules. During this process, water provides the reducing

power and the pigments capture the light energy.

The biochemical processes of photosynthesis may be divided into two

main groups: the light reactions (need light) and the dark reactions

(need no light).

In nut shell, during photosynthesis, after decomposition of water into

component elements, carbondioxide is reduced to give carbohydrate.

In green plants there are two photosynthesis systems, A and B. Both

differ in the type of chlorophyll present and in the chemicals using the

absorbed photon energy.

174

In higher plants and in cyanobacteria a photochemical reaction centre

and the light harvesting complex (antenna complex) associated with it

together form a photo system. There are two such photosystems,

photosystem-I and photosystem-II, embedded in the thylacoid

membrane and linked with each other through a thermochemical

bridge consisting of several electron carriers, particularly the mobile

plastoquinone (PQ) and plastocyanin (PC).

In photosystem-I (Photosynthesis system-A) the common strong

reducing species, REDA and the strong oxidising species, OXA are

formed, while in photosystem-II (photosynthesis system-B) a strong

oxidising agent, OXB but a weak reducing agent, REDB are obtained.

The function of OXB is to produce molecular oxygen. A manganese

complex, reduce OXB, which is regenerated by an another excited

chlorophyll molecule, for further use. In this redox reaction

manganese is converted into +2, +3 and +4 states.

REDA gives its electron to a carrier like ferrodoxin [Fe++

/Fe+++

] as a

result NADPH is formed which is a stable source for reduction of

carbon dioxide into carbohydrate.

3.10 CHECK YOUR PROGRESS : THE KEY

1(a) (i) Biochemical thermodynamics

energy change.

(ii) G

chemical potential

proceeds spontaneously

exergonic

175

gain in energy

endergonic

(iii) ATP

ATP

ATP

ATP

ATP and Pi

(b) (i) energy rich molecule

negatively charged oxygen

ATP

the repulsive the force.

high

(ii) DNA-polymerase

poly metrication

deoxyribonucleoside

triphosphates

Mg++

primer DNA.

(iii) Carbon di oxide

water

68 moles of ATP

2(a) (i) Life process

Chlorophyll

photosynthesis

(ii) porphyrin

Mg(II) ion

four nitrogen

176

porphyrin ring

square planar

(iii) conjugation

porphyrin

350-700 mm

photosynthetic pigments

(b) (i) solar energy

chemical energy

CO2

organic molecule

(ii) light reactions

dark reactions

(iii) (a) to capture photons with high efficiency and at a

reasonable metabolic cost and transfer them to a reaction

centre, and

(b) to convert solar energy into chemical energy.

177

M.Sc. (Final) Chemistry

PAPER –II : BIOINORGANIC, BIO ORGANIC & BIOPHYSICAL

CHEMISTRY

BLOCK – II

Unit-4 : Electron Transfer in Biology

Unit-5 : Biopolymer

Unit-6 : Biopolymer Characterisation

Author – Dr. Purushottam B. Chakrawarti

Dr. Aruna Chakrawarti

Editor - Dr. Anuradha Mishra

178

SUMMARY

Electron transfer involve redox reactions. Since removal of electrons

is oxidation and gain of electrons is reduction. The common metabolic

pathway for the channeling of electrons to oxygen is termed the electron

transport system. Most of the ATP used by animals is formed as the result of

the operation of this sequence of reactions. Metalloproteins, cytochromes

and nitrogease play important role in electron transfer.

Biopolymers include polysaccharides (cellulose, starch) proteins and

nucleic acids. The molecular characterisation of biopolymer involves modes

of linking of monomers and forces involved in their linking. Polypeptide or

protein – structure is determined in three steps : (i) The determination of

number of aminoacids (ii) The determination of order of linking of amino

acid and (iii) determination of their structure.

Characterisation of a biopolymer is a must for any worker dealing

with these. It is possible for the polymers to have the average molecular

weight but different molecular distribution. The molecular mass of a

polymer is expressed as number average mass ( M n) or weight average mass

( M w). M n is determined by employing methods using colligative

properties.

179

UNIT-4 ELECTRON TRANSFER IN BIOLOGY

Structure

4.1 Introduction

4.2 Objectives

4.3 Electron Transfer in Biology

4.3.1 Structure and Function of Metalloproteins in Electron

Transfer.

4.3.2 Cytochromes

4.3.3 Iron Sulphur Proteins Synthetic Models

4.4 Nitrogenase

4.4.1 Nitrogen-Fixation

4.4.2 Molybdenum Nitrogenase Other Nitrogenase Model Systems

4.4.3 Spectroscopic and Other Evidence

4.5 Diffraction Methods

4.5.1 Light Scattering

4.5.2 Low Angle X-ray Scattering

4.5.3 X-ray Diffraction and Photocorrelation Spectroscopy

4.5.4 ORD

4.6 Let Us Sum Up

4.7 Check Your Progress: The Key

180

4.1 INTRODUCTION

Removal of electrons is chemically defined as oxidation while

reduction is the gain of electrons i.e. transfer of electron in a system is a

redox system.

Electrons are tunneled from a variety of sources such as the six

oxidation reactions in the metabolism of glucose, to a single sequence of

oxidation-reduction reactions which passes them eventually to oxygen.

This common metabolic pathway for the channeling of electrons of

oxygen is termed the electron transport system, the respiratory chin, or the

cytochrome system. Most of the ATP used by animals is formed as the result

of the operation of this sequence of reactions. In certain reactions, the

electrons do go directly from the organic metabolite to oxygen rather than

through the electron transfer system. Most of the energy of these processes

appears as heat.

The details of the electron transfer system have proved to be difficult

to determine. A major reason is that the enzymes of the system occur in the

mitochondria in multi-enzyme aggregates which are called electron transport

particles. Apparently one unit of each necessary component is bound in a

specific spatial orientation with respect to the other components. This greatly

facilitates the transfer of electrons through the various steps of oxygen and

also the formation of ATP, giving in effect the greater efficiency of an

assembly-line process, Substrates such as malic acid or other oxidizable

181

compounds (as well of ADP, Pi and O2) continually enter the mitochondria,

and the reaction products, including ATP, continually leave the

mitochondria and are further metabolized in other parts of the cell.

This high degree of structural organization is fine for the cell, but it

makes of the task of unravelling the system a difficult one for the

biochemist. He can never be quite sure that a compound isolated from a

solution of broken electron transport particles actually was present in the

same form in the intact particle. Also, the properties of an isolated

component of the system may be considerably difference from those of the

component bound in place in the particle.

For these reasons some details of the electron transfer system remain

to be elucidated. Chief among these is the nature of the reactions in which

ATP is formed. As yet these cannot be given even as good guesses.

The electron transfer systems of different organisms, even of different

tissues of the same organism, may also differ somewhat in details, further

complicating the situation. Nevertheless, it is clear that the major

components of the electron transport systems are: (1) a diphosphorpyridine

nucleotide-containing enzyme, (2) a flavoprotein, (3) coenzyme Q, and (4) a

number of compounds called cytochromes. Figure 4.1 presents the currently

most popular view as to the nature of the sequential reactions which these

compounds undergo as they accept electrons and pass them on to the other

components.

Certain biological oxidations utilize a flavin rather than a pyridine

nucleotide as the coenzyme for the dehydrogenation of the reduced flavin

are passed back to DPN and then through the electron transfer system.

182

183

Figure 4.1: The Electron Transport System. The dashed line indicates

the flow of electrons.

In other, a notable example being the oxidation of succinic to fumaric acid-

one of the six oxidation reactions of glucose metabolism- the electrons pass

from the flavin to the cytochromes. DPN is thus bypassed. This eliminates

one of the sites of ATP formation. Such reactions yield only two ATP

molecules per molecule of original subtrate, rather than the common three

molecules of ATP for the entire system.

184

Figure 4.1 introduces a manner of representing chemical equations

which may be new to you. To clarify this notation, let us examine the first

reaction shown in the figure. This appears as:

oxidized

metabolite metabolite

H+

DPN+ DPNH

This equation would appear in the conventional manner as:

Metabolite + DPN+ oxidized metabolite + DPNH + H

+

Note also that the sites of formation of three ATP molecules are

indicated in the figure. Again it must be emphasized that the reactions

actually occurring at these points are unknown. Ultimately it will be

necessary to add new reactions at these sites, either directly in the course of

the electrons transfer or as side-reactions. Among the compounds currently

being considered as possible intermediates in the formation of ATP are a

phosphorylated histidine unit of a protein and a phosphorylated derivative of

Coenzyme Q, a quinone compound which can undergo readily reversible

reduction of the hydroquinone.

At present all that is actually clear is that the phosphorylation of ADP

(the addition of a phosphate group to ADP) is coupled with oxidation-

reduction reactions near the positions indicated. The synthesis of ATP as the

result of these reactions of the electron transport system is termed oxidative

phosporylation.

185

186

4.2 OBJECTIVES

The main aim of this unit is to discuss electron transfer (i.e. redox)

reactions in biological system. After going through this unit you would be

able to:

describe electron and function of metalloproteins in electron transfer,

discuss structure and function of metalloproteinase in electron

transfer,

understand important of cytochromes and iron sulphur proteins in

these reactions,

identified function of nitrogenase in nitrogen fixation, and

describe important of diffraction methods in the study of biological

systems.

4.3 ELECTRON TRANSFER IN BIOLOGY

As we know, oxidation is chemically defined as the removal of

electrons and reduction is the gain of electrons.

e-(electron)

Fe++

Fe+++

Substrate molecules are oxidized by removal of hydrogen by

dehydrogenases. The reduced dehydrogenases are then reoxidized by a

group of respiratory catalysts known as cytochrome system. The substrate is

thus oxidized by both processes. The reducing equivalents ultimately react

with molecular oxygen in the presence of cytochrome oxidase, the last

member of the cytochrome system.

The sequence of enzymes and carriers responsible for the transport of

187

reducing equivalents from substrates to molecular oxygen is known as

respiratory chain.

The respiratory chain is localised within mitchondaria. Formation of

ATP in the mito-chondria is the active area of research. Redoxpotential in

oxidation and reduction reactions, the free energy exchange is proportionate

to the tendency of reactants to donate or accept electrons. This is expressed

as an oxidation-reduction or redox potential.

4.3.1 Structure and Function of Metalloproteins in Electron Transfer

In biochemical reactions redox reactions are very important. In these

reactions some specific proteins function as biochemical catalysts (i.e.

enzymes). The mechanism of these proteins (enzymes) depends on the

presence of certain specific metal ions, i.e. they function in the form of

metalloenzyme (metallo-proteins). In these enzymes metal ions are present

as metal-chelates. The metal ions present in these metallo-proteins include

Fe(II), Cu(II), Mn(II), Zn(II), Mg(II) and Mo(III).

The metallo-proteins include in biochemical electron-transfer i.e.

redox reactions are characterized with the fact that a metal ion can form

coordination compounds of various oxidation potentials when coordinated

with different donor atoms. These metalloproteins are known as

oxidoreductase enzymes. These are classified in to five groups:

1. Oxidsaes:

(a) Enzymes that catalyze the removal of hydrogen from a substrate but

use only oxygen as a hydrogen acceptor to form water as a

AM2 2

1O

188

(Red) Oxidase

A

(Ox) H2O

hydrogen acceptor to form water as a reaction product (with the

exception of uricase and monoamine oxidase which form H2O2).

(b) They are conjugated proteins containing copper as prosthetic groups.

(i) Cytochrome oxidase:

(a) Cytochrome oxidase is a hemoprotein widely distributed in

plants and a animal tissues.

(b) It is the terminal component of respiratory chain found in

mitochondria.

(c) It is poisoned by cyanide and hydrogen sulfide.

(d) More recent studies show that 2 cytochromes are combined

with the same protein and the complex is known a cytochrome

aa3.

(e) Cytochrome aa3 contains 2 molecules of heme A, each having

one Fe atom, 2 atoms of Cu are also present which are

associated with the cytochrome oxidase activity.

(f) The terminal cytochrome aa3 is responsible for the final

combination of reducing equivalents with molecular oxygen.

(g) this enzyme system contains copper, a component of several

oxidase enzymes.

(h) It has a high affinity for oxygen.

(i) It is the only one in the chain which signifies the irreversible

reaction.

189

(j) It gives direction to the movement of reducing equivalents in

the respiratory chain and to the productions of ATP, to which it

is coupled.

(ii) Phenolase (tyrosinase, polyphenol oxidase, catechol oxidase):

(a) It is a copper containing enzyme.

(b) It converts monophenol to O-quinones.

(iii) Laccase:

(a) It is widely distributed in plants and animals.

(b) It converts P-hydroquinones to P-quinones.

(c) It also contains copper.

(iv) Ascorbic oxidase:

(a) It contains copper.

(b) It is found only in plants.

(v) Uricase:

(a) It also contains copper.

(b) It catalyzes the oxidation of uric acid to allantoin.

(vi) Monoamine oxidase:

(a) It is found in the mitochondria of several tissues.

(b) It oxidizes epinephrine and tyramine.

2. Aerobic dehydrogenases:

(a) They catalyze the removal of hydrogen from a substrate and use either

oxygen or artificial substances such a methylene blue as hydrogen

acceptor.

190

AH2 O AH MB H2O2

(Red) (Red) (Methylene blue)

Aerobic Aerobic Dehydrogenase Dehydrogenase

(Ox)A H2O2 (Ox)A MB-H2 O2

(b) H2O2 is formed as a product.

(c) They are flavoprotein enzymes having FMN (flavin mononucletide)

or FAD (Flavin ademine dinucleotide) as prosthetic groups.

(d) Many of the flavoprotein enzymes contain a metal for which they are

known as metalloflavoprotein enzymes.

(i) D-amino acid dehydrogenase (D-amino acid oxidase):

(a) It is an FAD-linked enzyme.

(b) It is found particularly in liver and in kidney.

(c) It catalyzes the oxidative deamination of the unnaturally (D-)

forms of amino acids.

(ii) L-amino acid dehydrogenase (L-amino acid oxidase):

(a) It is an FMN-linked enzyme.

(b) It is found in kidney.

(c) It catalyzes the oxidative demination of naturally occurring L-

amino acids.

(iii) Xanthine dehydrogenase (Xanthine oxidase):

(a) It occurs in milk and liver.

(b) In the liver, it converts purine bases to uric acid.

(c) It contains FAD as the prosthetic group.

191

(d) It is highly significant in the liver and kidneys of birds which

excrete uric as the end product of purine metabolism and also of

protein and amino acid catabolism.

(e) It is a metalloflavoprotein containing nonheme iron and

molybdenum.

(f) It also oxidizes all aldehydes.

(iv) Aldehyde dehydrogenase (aldehyde oxidase):

(a) It is an FAD-linked enzyme.

(b) It is present in pig and other mammalian liver.

(c) It is also a metalloflavoprotein containing nonheme iron and

molybdenum.

(d) It oxidizes aldehydes.

(v) Glucose oxidase:

(a) It is an FAD-linked enzyme.

(b) It is prepared from fungi.

(c) It is used in estimating glucose.

3. Anaerobic dehydrogenases:

(a) They catalyze the removal of hydrogen from a substrate but not able

to use oxygen as hydrogen acceptor.

(b) They transfer hydrogen from one substrate to another by oxidation-

reduction reaction not involving a respiratory chain.

AH2 Carrier BH2

(Red) (Ox) (Red)

Aerobic Dehydrogenase

A Carrier-H2 B

192

(Ox) (Red) (Ox) (Dehydrogenase (Dehydrogenase

specific for A) specific for B)

(c) They perform oxidation of metabolite utilizing several components of

a respiratory chain.

(i) Dehydrogenase dependent on Nicotinamide Conezymes:

(a) They are linked as coenzymes either to NAD (Nicotinamide

adenine dinucleotide) or to NADP (Nicotinamide adenine

dinucleotide phosphate).

(b) The coenzymes are reduced by the particular substrate of the

dehydrogenase and reoxidized by a suitable electron acceptor

and synthesized from the vitamin niacin (nicotinic acid and

nicotinamide).

(c) NADP-linked dehydrogenases catalyze-oxidoredutcion

reactions in glyolysis, the citric acid cycle and in the respiratory

chain of mitochondria.

(d) NAD-linked dehydrogenases are found in fatty acid and steroid

synthesis in the extramitochondria. They are also found in

hexose monophosphate shunt.

(e) Some nicotinamide coenzyme-dependent dehydrogenases

contain zinc, particularly alcohol, dehydrogenase from liver and

glyceradehyde-3-phosphate dehydrogenase from skeletal

muscle. The Zinc ions do not take part in the oxidation and

reduction.

(ii) Dehydrogenases dependent on Riboflavin Prosthetic Groups:

193

(a) Most of the riboflavin-linked anaerobic dehydrogenases are

concerned with electron transport in the respiratory chain.

(b) Succinate dehydrogenase, acyl-CoA dehydrogenase and

mitochondrial glycerol-3-phosphate dehydrogenase transfer

electrons directly from the substrate to the respiratory chain.

(c) In the dehydrogenation of reduced lipoate, an intermediate in

the oxidative decarboxylation of pyruvate and -Ketoglutarte,

the flavoprotein (FAD) due to the low redox protential acts as a

carrier of electrons from reduced lipoate of NAD+. The electron

transferring flavoprotein in an intermediary carrier of electrons

between acyl-CoA dehydrogenase and the respiratory chain.

4. Hydroperoxidases:

They utilize hydrogen peroxide as a substrate. Two enzymes fall into

this category (i) Peroxidase, (ii) Catalase.

(i) Peroxidase:

(a) It is found in milk and leukocytes and the prosthetic group is

protoheme.

(b) It catalyzes the reduction of hydrogen peroxide by the help of

ascorbic acid, quinones and cytochrome C which act as electron

acceptors. The reaction is complex but the overall reaction is as

follows:

H2O2 + AH2 Peroxidase 2H2O + A

(ii) Catalase:

(a) It is hemoprotein and found in blood and liver.

194

(b) It uses one molecule of H2O2 as a substrate electron donor and

another molecule as electron acceptor:

2H2O2 Catalase 2H2O + O2

(c) Its function is to destroy H2O2 formed by the action of aerobic

dehydrogenases.

5. Oxygenases:

They catalyze the incorporation of oxygen into a substrate molecule.

(i) Dioxygenases (Oxygentranferases, true oxygenases):

(a) They catalyze the incorporation of two atoms of oxygen (O2) into

the substrate:

A + O2 AO2

(b) Enzymes containing iron as a prosthetic group eg. homogentisate

dioxygenase, 3-hydroxyxanthranilate dioxygenase and enzymes

utilizing heme as a prosthetic group such as L-tryptophan

dioxygenase (tryptophan pyrrolase) from the liver.

(ii) Mono-oxygenase (Mixed function oxidases, Hydroxylases):

(a) They catalyze the incorporation of only one atom of the oxygen

molecule into a substrate. The other oxygen atom is reduced to

water. A co-substrate is necessary for this purpose:

A - H + O2 + BH2 A - OH + H2O + B

(b) Many of the enzymes involved in steroid synthesis are mono-

oxygenase using NADPH as a co-substrate. They are found

195

mainly in the endoplasmic reticulum (Microsomes) of the liver

and in mitochondria and the microsomes of the adrenal glands.

(c) They are also involved in the metabolism of many drugs by

hydroxylation. They are found in the microsomes of the liver

together with cytochrome P450 and cytochrome b5. The drugs

metabolized by this system are benzpyrine, aminopyrine, aniline,

morphine and benzphetamine. But phenobarbital induce the

formation of microsomal enzymes and of cytochrome P450.

196

4.3.2 Cytochromes

In the respiratory chain the pathway of electron from NADH to

oxygen involves many electron carriers, which remain tightly bound to

proteins of respiratory chain. These proteins are now known to be organized

into three enzyme complexes, each characterized by the electron carriers

with which each interacts. Since many of the electron carriers in the

respiratory chain absorb light and change colour, and since each of these

carriers has a characteristic absorption spectrum, their behavior even in a

crude mixture can be traced spectroscopically. These electron carriers were

discovered in 1925 as compounds capable of undergoing rapid oxidation and

reduction.

The compounds called cytochromes were probably the first

components of the electron transfer system to be associated with oxidation

reduction reactions. As the name cytochrome (cell-color) indicates, each of

these compounds is colored. Furthermore, the oxidized and reduced forms

absorb light differently, giving different absorption bands when viewed with

a spectroscope. In 1886 MacMunn observed that certain strong absorption

bands of a cell suspension appeared as the oxygen of the solution was used

up, and disappeared when oxygen was admitted to the system. This

suggested that the compound was alternately oxidized and reduced. The term

"cytochrome" was later coined for the substance involved, even though the

precise structure was unknown.

By examining cells and tissues with a spectroscope, three types of

cytochromes (cytochrome a, b and c) were identified (actually five

cytochromes a, b, c, c1 and a3 are involved is respiratory chain), although

such a grouping is not functionally important. Cytochromes are the most

197

important electron carriers of the respiratory chain, which are related with

each other by the presence of a bound heme group, whose iron atom changes

from ferric state (Fe3+

) to ferrous (Fe3+

) state when it accepts an electron.

The heme group of cytochromes consists of a porphyrin ring holding an iron

atom with its four nitrogen's. Similar porphyrin rings are found in

hemoglobin of blood in animals and in chlorophyll of green plants. The three

dimensional structure of cytochrome c, the most extensively studied of the

five cytochromes is shown in Figure 4.3

Figure 4.2 : The Prosthetic Group of Cytochrome-c

198

Figure 4.3

The cytochromes excepting cytochrome oxidase are anaerobic

dehydrogenases. They are involved as carriers of electrons from

flavoproteins to cytochrome oxidase in the respiratory chain.

Cytochrome C:

1. It has a molecular wt. of 13000.

2. The iron porphyrin group of cytochrome c is attached to protein more

firmly than in the hemoglobin.

3. It is quite stable to heat and acids.

4. The reduced form of cytochrome c is not autooxidizable.

5. The peptide chain of human heart cytochrome c contains 104 amino

acids. Acetylglycine is the N-terminal amino acid and glutamic acid is

the c-terminal amino acid. Two crystalline residues are located at

positions 14 and 17. The linkage of iron in heme occurs through the

imidazole nitrogen of histidine residue at position 17 in the peptide

chain.

199

Owing to the differences of structure of the different cytochromes,

they differ in reactivity, particularly in their ability to accept and to donate

electrons. One of these compounds, thought to be cytochrome-b, is capable

of accepting an electron from reduced coenzyme Q. The oxidized from of

cytochrome-b has a ferric ion at the center of the porphyrin system; the

reduced form a ferrous ion. The reduction of cytochrome-b involves only the

transfer of a single electron. Two of these molecules are therefore necessary

to complete the reoxidation of the coenzyme Q. Note that the hydrogens of

the reduced coenzyme Q are not transferred to cytochromes; they are

released as hydrogen ions to the medium.

An electron is next passed from reduced cytochrome-b to one

cytochrome after another with alternate oxidation and reduction of the iron

atom. Finally a cytochrome called cytochrome-a; or more commonly

cytochrome oxidase, is reached. This cytochrome is distinguished by its

ability to undergo direct oxidation by molecular oxygen, a property rarely

found in biological systems. As a class, enzymes catalyzing reactions

involving oxygen are termed oxidases. It has been estimated that as much as

95 percent of the oxygen utilized by cells reacts in this single process, the

oxidation of the reduced form of cytochrome oxidase to the oxidised form.

The reaction is not completely understood. In addition to be iron-porphyrin

portion prosthetic group, the enzyme cytochrome a3, conations a copper ion.

There are indications that the cupric ion receives an electron from the

ferrous-porphyrin portion of the enzyme, being reduced to cuprous ion, and

that it is the enzyme-bound cuprous ion which is oxidized in the final step by

molecular oxygen.

200

Whatever the details of the reaction mechanism, cytochrome oxidase

is an exceedingly important enzyme. Inactivation of this enzyme , as occurs

by its combination with carbon monoxide or cyanide ion in rather low

concentrations, leads to the rapid death of the cells of most organisms. Their

utilization of oxygen is prevented, in turn prohibiting the formation of ATP

in sufficient quantities to meet the energy demands.

Cytochromes are also found in the endoplasmic reticulum

(cytochromes P-450 and b5) plant cells, bacteria and yeast.

Cytrochromes P450 is considered the most versatile biocatalyst. It has

been shown by the use of 18O2 that one atom of oxygen enters R-OH and

one atom enters water. This dual fate of the oxygen is responsible for

naming of monooxygenases as "mixed-function oxidases". The reaction

catalyzed by cytochrome P450 is represented below:

RH + O2 R - OH + H2O Reduced cytochromie Oxidised

P450 Cytochrome P-450

They are present in large amount in liver. They are present mainly in

the membranes of the smooth endoplasmic reticulum in liver and most other

tissues. In the adrenal, they are found in mitochondria as well as in the

endoplasmic reticulum; the various hydroxylases present in that organ play

an important role in cholesterol and steroid biosynthesis. The mitochondrial

cytochrome P450 system differs from the microsomal system in that it uses

an NADPH-linked flavoprotein, adrenodoxin reducates, and a non-heme

iron-sulphur protein, adrenodoxin.

4.3.3 Iron Sulphur Proteins Synthetic Models

201

These are the second major family of electron carriers having 2 or 4

iron atoms bound to an equal number each of sulphur atoms and cystein side

chains (Fig. 4.4). There are more iron-sulphur proteins than the

cytochromes, but they are not as well characterized, since their detection

requires electron spin resonance (ESR) spectroscopy.

Ubiquinone or coenzyme Q : Ubiquinone is a small hydrophobic

molecule dissolved in the lipid bilayer and is the simplest of all electron

carries. It can pick up one or two electrons and also picks up one proten with

each electron that it carries. Ubiquinone belongs to a class of molecules

called quinones. The corresponding electron carrier in photosynthesis is

plastoquinone, which is almost identical. For simplicity both ubiquninone

and plastoquinones are referred to as quinone and abbreviated as Q.

Copper atoms and flavin : In addition to the above electron carriers,

there are two copper atoms and a flavin, which remain bound to the proteins

of the respiratory chain and serve as electron carriers. Flavin remains

associated with NADH dehydrogenase complex and is the first acceptor of

electron from NADH, while copper atoms remain associated with

cytochrome oxidase complex at the end of the electron transport chain.

202

Figure 4.4: The structure of two types of iron-sulphur centers (2 Fe2S

type and 4FeS4 type) each iron sulphur centre carries only one electron

although it has more than one iron atoms.

4.4 NITROGENASE

4.4.1 Nitrogen-Fixation

In nature there is constant interconversion between the various forms

of nitrogen. This inter-conversion is traced by nitrogen cycle. The

atmospheric nitrogen is fixed i.e. converted into chemical compound by two

paths: (1) Lightning discharges convert nitrogen and oxygen to nitric oxide,

which in turn is oxidized to NO2. The NO2 dissolves in rain water to form

nitrates and nitrites which are washed into the soil.

(2) Nitrogen fixing bacteria which li on the roots of leguminous

plants, such as clover, convert N2 to proteins and other nitrogen compounds.

In this form of fixation of nitrogen, symbiotic bacteria takes part. The

enzyme responsible for this fixation is nitrogenase.

Nitrogense during its activity uses its two proteins: one smaller and

the other larger. Smaller protein has molecular weight in between 57000 to

203

73000 and has Fe4S4 functional group. The larger protein is a 22 tetramer

with molecular weight in between 220000 to 240000. In addition to the Fe-S

group, it has molybdenum atom in the centre.

4.4.2 Molybdenum Nitrogenase & other Nitrogenase Model Systems

As has been mentioned, the larger protein present in the enzyme

nitrogenase is generally called molybdenum nitrogenase. Because, the active

centre for bonding with dinitrogen in it is molybdenum atom.

Molybdenum nitrogenase has two molybdenum atoms, approximately

thirty iron atoms and thirty sulphide ions. Probably Fe-S group functions as

the redox centre, It has been shown that molybdenum atom is the active site

for bonding of atmospheric N2. In its coordination sphere many sulphur

atoms are present at a distance - 235 pm. Other heavy atoms, e.g. iron are

present at approximately 270 pm. The last source of reducing power is

pyruvate, and electrons are transferred to nitrogenase through ferredoxin.

Probably, two Mo(III) atoms forming a cycle through Mo(VI), give six

electrons, necessary for reduction of N2. Alternately, as the enzyme has large

number of ferredoxine type groups, hence due to availability of exits for

electrons, molybdenum remains in +1 or +2 oxidation states and easily binds

with N2 and its intermediate reluctants (Fig. 4.5).

204

Fig. 4.5

4.4.3 Spectroscopic and Other Evidence

For the analysis and study of biological phenomenon various

instrumental methods are used. Among these methods most important are

spectroscopic methods, which include absorption spectroscopy, infrared

spectroscopy, NMR and ESR spectroscopy and x-ray spectroscopy. These

methods are used mainly for the investigation of structure of various

compounds and the mechanisms of different phenomenons. For example:-

(i) Cytochromes are coloured compounds, the oxidised and reduced forms

absorb light of different wave lengths. They give different absorption

bands when examined with a spectroscope. By examining cells and

tissues with a spectroscope three types of cytochromes, cytochrome a, b

and c have been identified.

(ii) Similarly x-ray crystallography indicated that the heme group of

cytochromes (cytochrome-c oxidase) consists of a porphyrin ring

205

holding iron atom with its four nitrogen (Fig. 4.2). It has been shown to

have 13 different sub units.

(iii) The electron spin resonance (ESR) spectroscopy indicates structures of

iron-sulphur proteins with formula 2Fe2S and 4Fe4S type (Fig . 4.4).

(iv) Spectroscopy (x-ray crystallography) has also indicated the structure of

nitrogenase enzyme. It has been shown to contain 2 molybdenum

atoms, 30 iron atoms and 30 sulphide ions, while Fe-S group functions

as the redox centre. In its coordination sphere many sulphur atoms are

present at a distance ~ 235 pm and iron atoms at a distance ~ 270 pm.

The last source of reducing power is pyruvate (Fig. 4.5).

Check Your Progress - 1

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) Common metabolic pathway for the............................to

oxygen is termed the................system or...............system.

(ii) The mechanism of enzyme proteins depends on the presence

of...................i.e. they function in the form of.............

(iii)The oxidoreductase enzyme are classified into following five

groups:

1. .............................

2. .............................

3. .............................

4. .............................

5. .............................

(b) (i) Cytochromes were probably first components of.................to be

associated with..............reactions.

206

(ii) Spectroscopic examination identified............ types of

cytochromes............and.............The heme group of cytochromes

consists of...............holiday an..............atom with its four................

(iii)Nitrogenase is the enzyme responsible for ..............

by.............present on the roots of..............

4.5 DIFFRACTION METHODS

For determination of structures of biological molecules diffraction

methods have proved quite useful. These include x-ray diffraction, neutron

diffraction and electron diffraction. Out of these diffraction methods x-ray

diffraction is the most generally applicable and most widely used of all

methods of structure determination and it is used for this purpose.

4.5.1 LIGHT SCATTERING AND DIFFRACTION METHODS

A crystal is a periodic three-dimensional array of atoms which acts as

a three-dimensional diffraction grating for X-rays of wavelength comparable

to the interatomic distances and interplanar spacings within it. When a beam

of X-rays passes through the crystal, the X-rays diffracted by atoms in

successive planes will mutually extinguish one another unless reflections

from adjacent planes are in phase. If the distance between successive planes

is d and the grazing angle of incidence , the path difference between X-

rays scattered from neighboring planes is 2d sin . The condition that X-rays

of wavelength shall be reflected by any family of planes is then given by

the Bragg relationship.

n = 2d sin

207

Where d is directly related to the dimensions of the unit cell and the

Miller indices of the reflecting planes. (The Miller indices of a family of

planes are the reciprocals of the fractional intercepts which the planes

nearest the origin makes with the crystallographic axes.) In essence, the

determination of the dimensions of the unit cell (the smallest repeating unit

from which the lattice can be built up by packing without any particular

orientation) involves measurement of the Bragg angles, allocation of the

correct indices and evaluation of the interplanar spacing.

The general principles of neutron diffraction by single crystals or

powders, which is used mainly for structure determinations, are similar to

those of X-rays diffraction. Neutrons produced by fission in a nuclear

reactor and slowed down to thermal velocities by passage through a

moderator have wavelengths of about 1.5 A, and reflection of the neutron

beam from a crystal of calcite or lead gives a beam of a small band of

wavelengths (not a strictly monochromatic beam). Even the strongest

neutron beams available are considerably weaker than an ordinary X-ray

beam used in crystallography, and larger crystal or samples are therefore

needed for neutron diffraction than for X-ray diffraction.

Despite these limitations, neutron diffraction has some very powerful

advantages. For diamagnetic species all the scattering is done by nuclei;

most of it is resonance scattering resulting from the formation and

immediate disintegration of unstable nucleus-neutron combinations (isotopes

which have high cross-sections for neutron capture must obviously be

avoided in neutron diffraction). The overall scattering factors of different

atoms vary over a factor of only about four, and in an erratic manner; 2H2,

12C,

14N and

16O have scattering factors which are about the same as those of

208

the heavy elements. Neutron diffraction is thus particularly useful for the

location of light atoms in structures where the X-ray scattering would be

dominated by heavy atoms. It is. in fact, common practice to determine the

positions of heavy atoms first by X-ray diffraction, and then to determine the

positions of light atoms by neutron diffraction.

The uses of electron diffraction are restricted to the study of gases and

vapours and thin films. Because of their negative charges, the scattering of

electrons is about a thousand times as effective as that of X-rays. Scattering

is by the potential filed of the species under study; for heavy element this

can be considered to reside at nuclear positions. Like X-rays, electrons are

scattered much more by heavy than by light atoms, and, as in X-ray

diffraction, it is impossible to locate hydrogen atoms accurately in the

presence of much heavier atoms.

4.5.2 Low Angle X-ray Scattering

When an X-ray beam is passed through matter, part of its energy is

lost by scattering and a part by absorption. Scattering of X-ray radiation

forms the basis for diffraction. In other words, the electrons in the atoms of

the matter absorb energy from X-rays and become excited producing

secondary radiation characteristic of the atoms.

All modern methods of obtaining diffractions patterns employ

monochromatic X-radiation. In the powder method the X-ray beam impinges

on a fine powder in which the particles are orientated at random; some,

however, are so placed as to satisfy the Bragg law for each value of d. The

reflections are recorded photographically on a thin cylindrical film in

circular camera. Reflections from planes with closely similar spacing but

209

different Miller indices are not separated adequately, however, and the

powder method is really suitable only for crystals having unit cells of fairly

high symmetry and, consequently, fewer possible values of d for a given

values of .

In the rotating crystal method the X-ray beam strikes a small crystal

which is slowly rotated about a vertical axis; the crystal is mounted so that

the axis of rotation is also one of the principal axis of the crystal. The angle

of incidence is thus continuously varied. When Bragg's law is satisfied, a

diffraction pattern is obtained and is recorded photographically or, for very

accurate work, by means of an ionization counter. The crystal is then

remounted so that all its important axes in turn are perpendicular to the X-

ray beam. For a compound with a large unit cell the closeness of the

reflections causes difficulties; these may be diminished by oscillation of the

crystal through a small angle instead of by rotation 360o, or by synchronized

movement of both crystal and film.

After the unit cell parameters have been obtained, the independently

measured density of the crystal allows the number of molecules in the unit

cell to be deduced. In some simple cases the systematic absence of certain

reflections corresponding to particular values of the Miller indices suffices

to determine the structure completely. Usually, however, it is necessary to

measure accurately the relative intensities of the reflections. The intensity of

reflections/from a particular plane is proportional to the square of a quantity

called the structure factor F, which is a function of the positions of the atoms

present and their scattering powers. These last depend on the numbers of

extra nuclear electrons for low-angle scattering, but decrease by calculable

amounts as increases. The problem is then to compare values of F

210

calculated for a postulated structure with those obtained by experiment, and

to 'refine' the postulated structure to give the best agreement between

observed and calculated structure factors.

4.5.3 X-ray Diffraction & Photo-correlation Spectroscopy

When an X-ray beam is passed through a substance, the electrons of

its atoms emit electromagnetic radiation in all directions like that of the

incident X-radiation. These scattered waves from the electrons are arranged

in the form of a crystal lattice. The interference of these waves causes

diffraction by the crystal plane. Thus each crystalline substance scatters the

X-ray in the form of its own diffraction pattern according to its atomic and

molecular structure. X-rays are electromagnetic waves having definite

wavelengths and the atoms are found in regular three-dimensional structure

in crystals. Therefore, X-radiations resolve the atoms found in a crystal and

the atoms of the crystals now scatter the X-rays, thus producing a diffraction

pattern in the form of spots. The diffraction pattern thus obtained is recorded

on a photographic plate. In the powder crystal diffraction spectrometer the

pattern on the photographic plate is recorded in the form of lines (Fig. 4.6).

211

4.6: Diagrammatic structure of powder crystal diffraction spectrometer.

X-ray diffraction spectroscopy is generally used to differentiate 'cis'

and 'trans' isomers and linkage isomers of a complex. The transmission

photographic method is useful to study the orientation of fibers and the

symmetry of a single crystal. Ionization spectroscopy is used to study

crystalline structures of molecules, while rotating spectroscopy is used to

determine the size of unit cells. Powder crystal spectroscopy is useful to

study cubic crystals.

4.5.4 Optical Rotatory Dispersion (ORD)

Most light as it occurs in nature is unpolarized and it consists of

different wavelengths and vibrates in many different planes. If the magnetic

field of light vibrates in a specific direction, then the light is said to be

linearly polarized. If the linearly polarized light contains additional

component of unpolarized light, it is partially polarized. If the electric field,

instead of oscillating in a plane, proceeds in the form of a helix around the

axis of propagation with a constant magnitude, then the light is referred to as

circularly polarize. Here, the electric field completes one revolution within

one wavelength. If the tip of the field rotates clockwise, then the light right

circularly polarised light (RCPL). If it rotates anticlockwise, then it is left

212

circularly polarized light (LCPL) (Figure 4.7). Elliptical polarization is the

intermediate condition between circular and linear polarizations. In other

words, linear and circular polarizations are two extremes of elliptical

polarization. Light can be polarized by scattering. Reflection, transmission,

selective absorption or double refraction.

Figure 4.7: Rotation of circularly polarized light.

When a beam of linearly polarized light passes through a

substance, the light remains linearly polarized but its plane of oscillation

rotates, i.e. its plane of vibration becomes changed in orientation. Such

substances are called optically active and the effect is called optical activity

or optical rotation. When looked towards the source, the rotation may be

clockwise and is called as right-handed rotation, ant the substance is called

dextrotatory or it may be counter clockwise called as left-handed rotation

and the substance is called as levorotatory (Figure 4.8). Most of the

biological molecules can rotate the plane polarized light so that they are

optically active. For example, most of the sugars are dextrotatory while the

proteins and amino acids as well as phospholipids are levorotatory. The

secondary and tertiary structures of proteins and DNA are asymmetric and

so they are optically active.

213

Dextrorotation Levorotation

Figure 4.8: Rotation of light by optically active molecule.

The optical activity of a substance is due to circular double refraction

of light, In other words, the substance has refractory index for right-

circularly polarized light, which is different from the refractive index for

left-polarized light. This means that the linear polarization of light is due to

two circular polarizations of the same amplitude but with opposite rotation

(circularly birefringence). The degree of rotation is proportional to the

differences between two refractive indices and length of light velocities so

that the emerging light beam will be out of phase with each other. Therefore,

the light is rotated by an angle to the original plane of polarization (Figure

4.9) and is dependent on wavelength. Thus at different wavelengths, the

rotation of light is different and this effect is called optical rotatory

dispersion (ORD).

Figure 4.9 : Rotation of polarized light from one plane to another.

The absorption of light by a molecule at a given frequency is different

in different directions. Therefore the molecule appears to have different

colours when viewed with two kinds of plane polarized light. This

214

phenomenon is called dichroism. If the wavelength of the plane polarized

light falls within the range of the absorption maxima of the medium, the rays

absorbed by the medium will prefer either left or right circularly polarized

rays. Therefore the transmitted rays have lesser intensity than the incident

rays. This differential absorption of circularly polarized light is called

circular dichroism (CD).

ORD and CD are closely related phenomena so that CD spectrum of a

sample can be derived from its ORD spectrum by mathematical calculations.

Uses:

ORD and CD spectra are useful in the study of conformational

changes in proteins and polypeptides. Proteins exhibit optical activity

as they are formed by L-amino acids which are arranged orderly as -

helices and possess asymmetric distribution of charges in tertiary

structure. Therefore, the general structure of protein molecules can be

obtained through the determination of its optical activity.

These methods are used to study enzyme-substrate complexes.

In combination with ribose, sugar, the bases in nucleic acids become

optically active with highest activity in helical structures. Therefore,

ORD/CD spectra give knowledge about structural conformation of

nucleic acids, their denaturation and their binding with proteins.

Check Your Progress - 2

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

215

(A)(i) A crystal is a periodic...............array of atoms which acts as a

.................for X-rays of wave length comparable to the...............

and ............ within it.

(ii) Bragg relationship is..............

(iii) When an X-ray beam is passed through matter part, of its energy is

lost by..............and a part by........... Scattering of X-ray radiation

forms the basis of..................

(iv) X-radiations resolve the atoms found in a crystal and the atoms of

the crystals now scatter ........, thus producing a ..........in the form

of............or........

(B)(i) Substance which rotate.............are called..............

(ii) At different................the................is different. This effect is

called.................

(iii) ORD spectra are useful in the study of............changes

in..........and...............

4.6 LET UP SUM UP

After going through this unit you would have achieved the objectives

stated at the start of this unit. Let us recall what we have discussed so for:

Removal of electron is chemically defined as oxidation and reduction

is gain of electrons. The common metabolic pathway for the

channeling of electrons to oxygen is termed the electron transport

system, the respiratory chain or the cytochrome system. Most of the

ATP used by animals is formed as the result of the operation of this

sequence of reaction.

216

The details of the electron transfer system have proved to be difficult

to determine. A major reason is that the enzyme of the system occur in

the mitochondria in multienzyme aggregates which are called electron

transport particles. This greatly facilitates the transfer of electrons

through the various steps to oxygen and also the formation of ATP,

giving in effect the greater efficiency of an assembly line process.

The phosphorylation of ADP is coupled with oxidation reduction

reactions near the positions mediated. The synthesis of ATP as the

result of these reactions of the electron transport system is termed

oxidative phosphrylation.

In biochemical reaction, redox reactions are catalyzed by a number of

proteins, called enzyme. These proteins function in the form of

metalloenzymes, since the mechanism of their enzyme activity

depends of the preserve of certain specific metallic ions. These

metalloproteinase are known as oxidoreductase enzymes, These are

classified into five groups;

1. Oxidase

2. Aerobic dehydrogenases

3. Anaerobic dehydragenases

4. Hydroperoxidases

5. Oxygenases

In the respiratory chain the pathway of electron from NADH to

oxygen involves many electron carriers, which remain tightly bound

to proteins of respiratory chain. These proteins are now known to be

organized into three enzyme complexes, each characterized by

217

electron carriers with which each interacts. Since many of the electron

carries in the respiratory chain absorb light and change colour, and

since each of these carriers has a characteristic absorption spectrum,

their behavior even in a crude mixture can be traced spectroscopically.

These compounds are called cytochromes and probably are the first

components of the electron transfer system to be associated with

oxidation reduction reactions.

By examining cells and tissues with a spectroscope three types of

cytochromes (cytochrome a, b and c) were identified.

The heme group of cytochromes consists of a porphyrin ring holding

an iron atom with its four notrogens.

Owing to the difference of the different cytochromes, they differ in

reactivity, particularly in their ability to accept and to donate

electrons.

Cytochrome oxidase is distinguished by its ability to undergo direct

oxidation by molecular oxygen, a property rarely found in biological

systems.

In addition to the iron porphyry prosthetic group cytochrome

3contains a copper ion. There are indications that the cupric ion

receives an electron from the ferrous porphyrin portion of the enzyme,

being to cuprous ion and that it is the enzyme bound cuprous ion

which is oxidized in the final step by molecular oxygen.

218

Iron sulphur proteins are the second major family of electron carriers

having 2 or 4 iron atoms bound to an equal number each of sulphur

atoms and systein side chains. Two types of iron sulphur centres have

been identified (2Fe2S type and 4Fe4S type), each iron sulphur centre

carries only one electron, although it has more than one iron atom.

The atmospheric nitrogen is fixed i.e. converted into chemical

compound by nitrogen fixing bacteria which lie on the roots of

leguminous plants, such as clover, convert N2 to proteins and other

nitrogen compounds. In this form of fixation of nitrogen, symbiotic

bacteria take part; the enzyme responsible for this fixation is

nitrogenase.

Nitrogenase during its activity uses its two proteins, one smaller with

molecular weight in between 57000-73000 and Fe4S4 functional

group, and the other larger, with molecular weight 22000-240000 and

is a 2 2 tetramer. It has two molybdenum atoms, approximately 30

iron atoms and 30 sulplide ions. Probably Fe-S group functions as the

redox centre and molybdenum atom is the active site for bonding of

atmospheric N2.

For determination of structure of biological molecular, diffraction

methods are quite useful, particularly, X-ray diffraction is the most

generally applicable and most widely used of all methods of structure

determination.

A crystal is a periodic three dimensional array of atoms which acts as

a three dimensional diffraction gratting for X-rays of wave length

219

comparable to the interatomic distances and interplanar spacings

within it.

All modern methods of obtaining diffraction patterns employ

monochromatic X-radiation, when an X-ray beam is passed through a

substance, the electrons of its atoms emit electromagnetic radiation in

all directions like that of the incident X-radiation. These scattered

waves from the electrons are arranged in the form of a crystal lattice.

Therefore X-radiations resolve the atoms found in a crystal and the

atoms of the crystal now scatter the X-rays, thus producing a

diffraction pattern in the form of spots/lines and is recorded on a

photographic plate.

ORD is useful in the study of conformational changes in proteins and

polypeptides, and the method is used to study enzyme substrate

complexes.

The optical activity of a substance is due to circular double refraction

of light. This means that the linear polarization of light is due to two

circular (left and right) polarizations of the same amplitude but with

opposite rotation. Therefore the light is rotated by an angle to the

original plane of polarization and is dependent on wave length. Thus

at different wavelength, the rotation of light is different and this effect

is called optical rotatory dispersion (ORD).

4.7 CHECK YOUR PROGRESS: THE KEY

1(a)(i) channeling of electrons.

electron transport

220

cytochrome

(ii) specific metal ion

metallo enzyme.

(iii) 1. Oxidase

2. Aerobic dehydrogenases

3. Anerobic dehydrogenases

4. Hydroperoxidase

5. Oxygenase

(b)(i) electron transfer system

oxidation – reduction

(ii) three

a, b and c

porphyrin ring

iron

nitrogens.

(iii) fixation of nitrogen

symbiotic bacteria

leguminous plants

2(a)(i) three dimensional array

three dimensional grating

interatomic distance

interplanar spacings

(ii) n = 2d sin

221

(iii) scattering

absorption

diffraction

(iv) the X-ray

diffraction pattern

spots

lines

(b)(i) plane of polarized light.

optically active

(ii) wave lengths

rotation of light

optical rotatory dispersion (ORD)

(iii) conformational changes.

proteins

polypeptides.

222

UNIT-5 BIOPOLYMER

Structure

5.1 Introduction

5.2 Objectives

5.3 Chain Configuration of Macromolecules

5.3.1 Calculation of Average Dimensions for Various Chain

Structures.

5.4 Polypeptide and Protein Structures

5.4.1 Forces Involved in Biopolymer Interaction.

5.4.2 Electrostatic Charges and Molecular Expansion of Hydrophobic

Forces.

5.4.3 Dispersion Force Interactions.

5.4.4 Multiple Equilibria and various Types of Binding process in

Biological system.

5.5 Hydrogen Ion Titration Curves.

5.6 Let Us Sum Up

5.7 Check Your Progress: The Key

223

5.1 INTRODUCTION

The term 'Polymer' is a Greek word meaning combination of many

molecules ('Poly' means many and 'mer' means part, molecules or a unit).

Hence polymerization is defined as the combination of many molecules and

polymer is a compound of high molecular mass formed by the combination

of large number of small molecules. The small molecules which form the

repeating units are called monomer units. For example, polythene is a

polymer obtained by the combination of many unit of ethane or ethylene.

Thus in polythene, ethylene is the monomer.

Such polymers which are obtained only from one type of monomer

molecules are called homopolymers. There are many examples of polymers

which are obtained by the polymerisation of two different type of monomer

units. Nylon and terylene are such examples. Such polymer in which the

repeat units are made up of two different monomer are called copolymers or

mixed polymers. Polymers are also called macromolecules as they are the

big or giant molecules. Thus-

Monomers are the simple molecules which combine to form a

polymer.

224

Homopolymers are made up of only one kind of monomer units.

Copolymers are made up of two kinds of monomer units.

Broadly speaking, polymers have been classified into two categories

namely natural and synthetic polymers. There are many common examples

of natural polymers. Starch, cellulose, proteins, nucleic acids and natural

rubber are some of the examples of natural polymers. Natural are polymers -

often called biopolymers.

Starch and cellulose are made up of glucose units. Cellulose is made

by plants from glucose produced during photosynthesis. Proteins are the

polymers of -amino acids. Nucleic acids are polymer of nucleotides.

Wool, natural silk, hair, leather and skin contain proteins natural rubber is

made up of isoprene units (2-Methyl -1, 3-butadiene).

Carbohydrates, composed of carbon, hydrogen, and oxygen, are the

main source of cellular energy and are also important structural components

of cell walls, and intercellular materials. Carbohydrates are classified

according to the number of monomers they contain into monosaccharides

(e.g. glucose, fructose), disaccharides (e.g. sucrose, lactose) and

polysaccharides (e.g. starch, cellulose etc.).

Polysaccharides result from the condensation of many hexose

monomers, with a corresponding loss of water molecules. Their formula is

225

(C6H10O5)n. Upon hydrolysis they yield molecules of simple sugars. The

most important polysaccharides in living organisms are starch and glycogen,

which are reserve substances in plant cell and animal cells, respectively, and

cellulose, the most important structural component of the wall. These three

substances are all polymers of glucose molecules, but differ in the way they

are joined together. Carbohydrates serve as sources of energy or play a

structural role (e.g. in cell walls).

Another class of compounds, essential for living beings is proteins.

Thousands of different proteins go to the making up of a living cell. Proteins

are polyamides formed from amino acids; protein is obtained as a result of

polymerization of amino-acids. Protein, a long-chain polymer, sometimes

cross-linked, is composed of 20-1000 amino-acids in a highly organized

arrangement.

There are 20 commonly occurring amino acids in proteins and 6 are

found in special tissues. The amino acids differ with respect to the nature of

their side chain groups R. The properties of amino acids side chains

determine the properties of the proteins they constitute. The human bodies

can synthesis 10 out of the 20 amino acids found in proteins. Others must be

supplied in the diet and these are called essential amino acids. The proteins

in corn, rice and wheat have low lysine, tryptophan and threonine content.

These are supplemented by alternative protein-rich diets like pulses, etc.

Lack of essential amino acids in diet can cause diseases such as

Kwashiorkar.

226

All living organisms contain DNA and RNA. The genetic information

contained in the DNA is transcribed into RNA, which in turns is translated

into protein; this series of events is often referred to as the central dogma.

Nucleic acids are linear polymers of nucleotides linked together by

phosphodiester bonds. The nucleotide monomers result from the covalent

bonding of a phosphate and a base to a pentose moiety. The pentose is ribose

in RNA and deoxyribose in DNA. The bases found in DNA are thymine (T)

and cytosine (C), which are pyrimidines, and adenine (A) and guanine (G),

which are purines. RNA contains Uracil (U) instead of thymine. A

nucleotide without its phosphate group is called a nucleoside. In addition to

their role as nucleic acid constituents, nucleotides also have a major role in

the storage and transfer of chemical energy.

All the genetic information of a living organism is stored in its linear

sequence of the four bases. Although DNA base composition varies from

one species to another, the amount of adenine always equals the amount of

thymine (A = T), and the amounts of cytosine and guanine are also equal (C

= G).

A DNA molecule is composed of two anti parallel polynucleotide

chains that form a double helix around a central axis. The bases are stacked

inside the helix in a plane perpendicular to its axis, and the two strands are

held together by hydrogen bonds established between the base pairs. The

only pairs that occur are AT, held together by two hydrogen bonds and GC,

and held together by three hydrogen bonds. The latter pair is the more stable.

The pairing properties of the bases are such that, whatever the axial

227

sequence on one strand may be, the sequence on the other strand must be

exactly complementary to it.

If the two strands of a DNA molecule are denatured (separated) by

physical or chemical treatments, they can subsequently reanneal as a

consequence of their nucleotide base-pairing properties. Renaturation studies

have led to the discovery of repeated sequences in eukaryotic DNA, similar

hybridization studies have provided a powerful method for characterizing

RNA molecules, which hybridize only to the DNA from which they were

transcribed.

The three major classes of RNA are messenger, transfer and

ribosomal all of which are involved in protein synthesis. Although each

RNA molecule consists of only a single polynucleotide chain, the chain

folds upon itself to make a more compact structure having such secondary

structure characteristics as hairpin loops. These structures may have

important consequences.

5.2 OBJECTIVES

The main aim of this unit is to study structures of biopolymer. After

going through this unit you would be able to:

describe chain configuration of macromolecules and calculate average

dimension of these chain structures,

discuss structures of proteins and polypeptides,

understand integrations of biopolymers and explain forces involved in

biopolymer interactions,

discuss electrostatic charge and molecular expansion of hydrophobic

forces, and

228

describe multiple equilibria and various types of binding processes in

biological system.

5.3 CHAIN CONFIGURATION OF MACROMOLECULES

Polymers may be linear, branched chain or have three dimensional

network (cross linked) structures. However, most of the biopolymers are

linear polymers. In linear chain polymers, monomer units are linked together

linearly and form long straight chains.

If A is the monomeric unit then linear chain polymers may be

represented as given in Fig. 5.1

~~A-A-A-A-A-A-A-A~~ OR

Fig. 5.1: Linear chain polymer

The polymeric chains of the above type are put over one another to

give a well packed structure. Due to better packing on the monomeric units,

the linear polymers have high densities, high tensile strength and high

melting points.

Besides the long chains of monomeric units, there are some side or

crossed link chains also which can be of different lengths. Branched chain

polymers are irregularly packed due to

which they have lower tensile strength,

lower density and lower m. p. as

229

compared to the linear chain polymers (Fig. 5.2).

Fig. 5.2 (a) Branched chain polymer (b) Cross-linked Polymer

These linear polymers are formed by joining of monomeric units

together to form polymeric chains. The polymerization process generally

takes place through condensation, precisely, through the stepwise

intermolecular condensation process with the loss of molecules of generally

H2O (or HCl, NH3, etc.) e.g. just as, in Fig. 5.3, two monosaccharides can

link to form a disaccharide, additional monosaccharide can be linked on two

give polysaccharide. For example, at either end of the maltose unit to

additional glucose can link to give long, nearly infinite, chains of glucose

(Fig. 5.4)

D-Glucose + D-fructose = sucrose

230

Fig. 5.3

Fig. 5.4

The huge linear polymer which is formed is one of the forms of

starch, which represents a reserve energy store for plants. However not all

starch has this relatively simple structure. Chemical studies on the

degradations of starch indicate that there are two types of starch, one, called

amylose, composed of the end-to-end linkages of glucose units, and a

second, called amylopectin, which contains both end-to-end linkages and

side-chain linkages, resulting from joining CH2OH groups to other glucose

units. When this occurs, branches in the chain and formed. In the branched

form of starch, one CH2OH unit out of 20 or 30 glucose units takes part in

the branching.

Similarly, in proteins, amino acids condense with the amine end of

one amino acid and the carboxyl end of the another, so as to split out water,

forms the peptide link, NH-CO. Further condensation polymerisation

produce longer chains (Fig. 5.5); or in

231

(a) Peptide Link (b) Protein Helix

Fig. 5.5: Formation of Protein Chain.

nucleic acid, RNA is a polymer composed of sugar (ribose) ring hooked

together through phosphate linkage (Fig. 5.6)

Fig. 5.6: RNA Polymer.

5.3.1 Calculation of Average Dimensions for various chain structures

232

Polymer chain interaction may result in an ordering of chains in

crystalline or amorphous phase. The small changes in size and distribution

of these ordered regions may bring about differences in the physical and

mechanical properties in different samples of polymers.

It is a difficult task to characterize cross-linked structures. Sometimes

solution techniques are used where uncross link polymers are dissolved.

True cross-linked polymer networks are of infinite size with many cross-

linked sites per chain forming a three-dimensional network of great

complexity.

Molecular Size

The molecular size can be determined by degree of polymerization

radius of gyration, hydrodynamic volume etc.

Degree of Plymerization (D.P.)

In a polymer, the repeating atomic grouping usually equivalent to the

monomer, are the starting materials for polymer.

nCH2=CH2CH2-CH2-CH2-CH2-or CH2(-CH2-CH2-)nCH2

n the number of repeating units in a macromolecule is called the

degree of polymerization. It can be obtained by dividing the molecular

weight of the macromolecule by the molecule weight of the monomer. It can

be directly determined from the intrinsic viscosity data as follows:

aPDK (9.2)

233

Where K and a are constants, and P is the degree of polymerization, a

varies from solvent to solvent and its value ranges between 0.5 and 1.6. The

value of varies from a few units to 10,000 and more.

Further, the root mean square end distance gives the dimension of the

molecule. This can be determined by the viscosity or light scattering of

dilute solutions.

Light Scattering

Doty and Steiner have obtained the solution to the equation between

( h-2

) 2

1

and anisotropy (Z) where ( h-2

) 2

1

is the root mean-square end to end

distance of a chain and Z is the asyminetry coefficient given by I90- 901I B

where = 90 .B They also prepared tables giving the value of ( h-2

) 2

1

and

(Z). Now (Z) is experimentally determined, so ( h-2

) 2

1

can be found from the

tables. We also know that for a Gaussian coil, R the radius of gyration is

given by:

2R = 2

6

1 h

Zim Method

In this method 2R is directly calculated. The slope of the curve ( R ) or

So is determined at C = 0 and = 0. Then for a Gaussian coil:

MSh

o22

2

8

9

Where is the wave length of the incident light. Thus the coil size

will depend on the solvent. It would be better if measurements are taken in

234

solvents (when the solution behaves as ideal solutions). Thus knowing , So,

M the value of 2h can be calculated.

Viscosity Method

The value of 2

1

2h can also be obtained by viscosity measurements:

M

hKo

2

3

2

Where k is the universal constant and is equal to 2.84 x 1023

. It was

observed that the values of h2 obtained according to above equation do not

coincide with those obtained by other methods. As a matter of fact the

values obtained solvents do not agree. The value of an in Equation 94

corresponds to 0.5. Tager has concluded that it is due to the selective

adsorption of the solvent by the coil, e.g. polystyrenes coil selectively

adsorbs benzene from a benzene methanol mixture. Therefore A2=0 near the

coil but it is not so inside the coil.

The dimension of a macromolecule in a solution is actually the size of

the effective sphere which depends on the quality of the solvent. In

polyvinyl napthalene fraction, the segments of polymer chain occupy only

1.5 percent of the coil volume and the remaining volume is occupied by the

solvent.

Molecule Weight Distribution (MWD)

It is possible for the polymers to have the same average molecular

weight but different MWD. MWD has been found to be an important

variable and contributes to physical properties like adhesion, toughness,

tensile strength, brittleness, gas permeability and stress crack resistance

235

Size-exclusion chromatography is used for the study of MWD. Certain

anionic polymerization processes produce narrow MWD, e.g. polystyrene,

on anionical polymerization can give .02.1/ nw MMd The value of d per

step growth polymerization is 1.5-2.0, for radical polymerization 2.0-5.0 and

for insertion polymerization 5.25. A log-normal distribution is found for

polyethylene and polypropylene.

5.4 POLYPEPTIDE AND PROTEIN STRUCTURES

As has been mentioned all proteins are polymers and consists of a

large number of simple building units called amino acids. These acids have

already been mentioned as bi-functional molecules which contain both

amine groups and a carboxyl group. Although 26 amino acids have been

found in nature, only 20 occur regularly in proteins.

236

Fig. 5.7: Chemical structure of the twenty amino acids classified acidic,

basic, neutral polar, and neutral non-polar. The structures below the

conserved amino and carboxyl group are the R side chains.

Because of the simultaneous presence of acidic (carboxyl) and basic

(amino) groups, amino acids can have both positive and negative charges

and are therefore amphoteric molecules or zwitterions.

The ionized form of an ammo acid is:

H

| +H3NCCOO

-

|

237

R

Figure 5.7 shows the structure of the 20 amino acids that are coded in

biological systems. Of these, two are acidic, aspartic acid and glutamic acid

(D and E in a one-letter nomenclature system that is sometimes used); there

are basic, lysine (L), arginine (R), and histidine (H); seven are neutral and at

the same time polar (i.e., hydrophilic), serine (S), threonine (T), tyrosine

(Y), tryptophan (W), asparagine (N), glutamine (Q), and cysteine (C); and

eight are neutral non-polar (i.e., hydrophobic), glycine (G), alanine (A),

valine (V), leucine (L), isoleucine (I), phen ylalanine (F), proline (P), and

methionine (M). Note that two of amino acids (Met and Cys) contain a

sulfur atom. Between two cysteines a covalent disulfide bridge (-S-S-) can

easily be formed because the H atoms of the –SH groups can be removed.

The names of amino acids are usually abbreviated by using the first three

letters of their names.

The condensation of amino acids to form a protein molecule occurs in

such a way that the acidic group of one amino acid combines with the basic

group of the adjoining one, with the simultaneous loss of one molecule of

water.

The linkage -NH-CO- is known as the peptide linkage or peptide bond

(Fig. 5.8). The formed molecule preserves its amphoteric character, since an

acidic group is always at one end and a basic group is at the other, in

addition to side chains that can be basic (Lys, Arg, His) or acidic (Asp, Glu).

A combination of two amino acids is a dipeptide; of three, a tripeptide.

When a few amino acids are linked together; the structure is an oligopeptide.

A polypeptide consists of many (some even 1000 or more) amino acids. In

238

the linear polymers there is always an amino (N-terminal amino acid) and a

carboxyl group (C-terminal amino acid) at the ends.

The distance between two peptide links is about 0.35 nm. A protein

with a molecular weight of 30,000 consisting of 300 amino acid residues, if

fully extended, should have a length of 100 nm, a width of 1.0 nm, and a

thickness of 0.46 nm.

It is important to stress that the properties of proteins vary

considerably. The side chains of the 20 amino acids have different chemical

properties, and the number of different molecules possible by changing the

linear sequence of amino acids is enormous. Much more diversity can be

obtained in the chemical properties of proteins than in those of nucleic acids,

which with their four component monomers are simple in comparison to

proteins.

Fig. 5.8: (a) Formation of a peptide bond.

(b) Polypeptide Chain.

5.4.1 Determination of Polypeptide Structure

239

For determination of structure of a polypeptide chain it is necessary to

find out:

(1) How many amino acids are present in the polypeptide chain and,

(2) What is the order of linking of amino acids in the chain.

Thus, this involves - (a) analysis of amino acids in the given

polypeptide chain and (b) determination of the order of amino acids present

in the polypeptide chain.

(a) Analysis of Amino Acids

For this, the given polypeptide (or the protein) is hydrolyzed with 6N

hydrochloric, so that the amino acids present in the chain are separated apart.

Exception is tryptophan which is not isolated, but is destroyed. Hydrolysis

with an alkali gives a raceme mixture, hence not used:

O

||

CH2NC2CNHC.NHCH.COOH

| | N2

CH3 CH2C6H5

- +

CIH3NCH2COOH+CIH3N.CH.COOH+CIH3N.CH.COOH

| |

CH3 CH2C6H5

GlycineHydrochloride Alanine Phenyl Alanine

Hydrochloride Hydrochloride

Now, isolation and identification of structure of each of the amino

acid is done using the following techniques:

240

(i) Ion Exchange Chromatography,

(ii) pH dependent precipitation, and

(iii) Electrophoresis

(b) Determination of the order of amino acids present in the

polypeptide chain

After knowing the number of amino acids, the sequence of amino

acids residue along the peptide chain is determined, using the technique

known as, 'End group Analysis'.

As a matter of fact, this is a difficult task, because the same amino

acids can link differently e.g. in a dipeptide containing glycine and alanine,

these two amino acids can link in two different ways:

CH3

|

(1) H2N.CH2.CONH.CH.COOH

CH3

|

(2) H2N.CH.CONH.CH2.COOH

In structure (1) N-terminal is glycine an C-terminal is alanine, while

in structure, (2) N-terminal is alanine and C-terminal is glycine.

So, for end-group analysis, analysis of the amino terminal and the

carboxylic terminal of amino acid of the polypeptide chain is done one by

one using the various well known standard methods.

I. Amino-End-Degradation

241

For determination of the amino terminal, the following amino-end-

degradation methods are used:

1. Sanger's Method (1945)

In this method the free amino group of a polypeptide chain is allowed

to react with Sanger's reagent, 2.4 di-nitro fluoro bezcene (DNFB), this gives

dinitrophenyl derivative of the polypeptide. This is then hydrolyzed with

acid, so that peptide bond breaks at the point where N-terminal of an amino

acid is linked with polypeptide residue. After the hydrolysis, the coloured

dinitro phenyl derivative is obtained, which is identified using thin layer

chromatographic (TLC) technique.

2. Adman's Method

In this method, the polypeptide is reacted with Adman's reagent,

phenylisothiocyanate (C6H5N = C = S). The reactio takes place in the

following steps:

C6H5N=C=S+H2N.CH.CONH.CH.CONH.CH.CO

| | |

242

R1 R2 R3

Phenyl isothicyanate

(Adman's Reagent)

S H

|| |

C6H5NH.C.N.CH.CONH.CH.CONH.CH.CO.

| | |

R1 R2 R3

N-Phenyl Thiocarbamil Pe pti de

C6H5 – N = C = S + HOOC – CH – NH2

|

R

The amino acid obtained in the end of the reaction is identified.

3. Densil's Method :

This method resembles sanger method but is more efective and

sensitive. The reagent used in this reaction is N-N dimethyl amino

naphthaline – 5 – Sulphonyl chloride (Densil Chloride). The reaction follows

the following steps :

243

Densil derivative of amino acid Mixture of amino acids

Densil derivative of the amino acid is identified using TLC method.

4. Enzymic Method

The enzyme amino peptidase reacting with a mixture of amino acids

isolates, one by one, an amino acid, which can be indentified accordingly.

II. Carboxyl End Degradation

The methods used for the purpose are as follows:

1. Schlack and Kumpf Method

In this method, the first step is protection of amino group by its

benzoylation. Then, in the second step, C-terminal amino acid is converted

into thiohydantoin. In the end step, using Adman's method it is hydrolysed :

244

The amino acid obtained in the end of the reaction is identified. The

process is further repeated with the degraded polypeptide.

2. Reduction Method

In this method the carboxyl terminal is reduced by lithium aluminum

hydride into a primary alcohol. After the hydrolysis C-terminal

amino acid gives an amino alcohol, which is identified. The process is

further repeated with the degraded polypeptide.

3. Hydrazinolysis (1956)

In this method the polypeptide is reacted (heate) with hydrazine, thus,

leaving the carboxy terminal, all the amino acids are converted into amino

245

acid hydrazide. After removing the free amino acids, the carboxy terminal is

identified :

4. Enzyme Method

Similar to aminopeptidase, carboxypeptidase enzyme is capable of

releasing all C-terminals, which can be indentified accordingly.

5.4.2 Structure of Proteins

As described earlier proteins are the polymers containing large

number of amino acids joined to each other by peptide bonds. For

establishing the structure of a protein molecule we will have to answer the

following questions.

(i) The nature of amino acids.

(ii) The number of each particular amino acid present in one molecule of

the protein.

(iii) The sequence in which the various different amino acids are arranged

in the molecule.

(iv) The shape of the peptide chain, i.e. whether it is linear, cyclic,

branched or arranged in the form of helix.

(v) The forces with which the individual peptide chains are held together.

246

(vi) The way in which the individual peptide chains are arranged in

definite manner to a macromolecule of an individual shape (folded,

refolded).

(vii) The number of peptide chains and their arrangement in the natural

protein.

The first three points constitute the primary structure, the point (iv)

constitutes the secondary structure, the points (v) and (vi) constitute the

tertiary structure; and the point (vii) constitutes the quaternary structure of

the protein molecule. The secondary or higher structures of proteins can

accurately be determined only by X-ray analysis; although other physical

methods like viscosity, light scattering, rotatory dispersion, etc, also provide

useful information.

(a) Primary Structure: Primary structure of a protein refers to the

number, nature and sequence of amino acid residues along the peptide

chains. The determination of the complex primary structure can be divided

into a number of following regular stages :

(i) Purification.

(ii) Determination of amino-acids composition. The protein is hydrolyzed

by means of acid (6, M, HCl, 110o), alkali [2NBa(OH)2, 110)

o or enzyme

(peptidase) to its constituent amino acids which are separated and identified

by means of reaction with ninhydrin. The quantity of each amino acid may

be determined by isotopic dilution method which consists in adding a known

amount of a C14

-labelled variety of the amino acid, whose analysis is sought,

to the mixture. The amount of each of the amino acids present in the

hydrolysate may also be determined by means of biological assays.

247

By knowing the relative amount of each of the amino acids present in

the molecule, the empirical formula in terms of the amino acids of the

protein can be deduced.

(iii) End-group determination: The N-terminal and the C-terminal

amino acids of a polypeptide chain can be determined one by one by the

various standard well-known methods. Thus the sequence of the amino acids

present in the polypeptide chain (s) is established.

Thus, the primary structure is the sequence of amino acids, which

form a chain connected by peptide bonds. The amino acid sequence of a

protein determines the higher levels of structure of the molecule. The

biological importance of the amino acid sequence is exemplified by the

human hereditary disease sickle-cell anemia. In which profound biological

changes are produced by a single amino acid change in the hemoglobin

molecule.

For example, hemoglobin, the molecule in blood that carries oxygen

consists of 574 amino acid units. Changing one specific amino acid in the

sequence results in defective hemoglobin found in patients suffering from

sickle cell anemia.

Normal Hemoglobin -Val-His-Leu-Thr-Pro-Glu-Glu-Lys-

Sickle cell hemoglobin -Val-His-Leu-Thr-Pro-Val-Glu-Lys-

Another example illustrating that fact that a small variation in the

primary structure can alter the physiological activity of a peptide or protein

is the structure of oxytocin and vasopressin.

248

The above two structures are quite similar with a variation in only the

third and eight positions (represented by black letters) of the peptide chain.

These changes alter the activities of the two hormones.

Oxytocin causes contraction of the smooth muscles of the uterus and

portions of the gastrointestinal tract, while vasopressin causes a constriction

of the peripheral body vessels and a decrease in the secretion of urine.

(b) Secondary Structure: The secondary structure is the spatial

arrangement of amino acids that are close to each other in the peptide chain.

Some regions may display a rod-shaped structure, the -helix (called alpha

because it was the first structure deduced by Pauling and Corey in the early

1950s). In an -helix the peptide chain is coiled around an imaginary

cylinder (Fig. 5.9 (a)) and stabilized by hydrogen bonds between the amino

group of an amino acid and the carboxyl group of the amino acid situated

four residues ahead in the same polypeptide chain. The protein -helix has

3.6 amino acids per turn. In a -pleated sheet (Fig. 5.9(b) the amino acids

adopt the conformation of a sheet of pleated paper, and the structure is

stabilized by hydrogen bonds between the amino and carboxyl groups in

different polypeptide strands. Other segments of the protein are not highly

cross-linked and adopt a random coil configuration (Fig. 5.9(c). This is

partly because certain amino acids, such as praline, tend to disrupt helical

structure.

249

Fig. 5.9: Representations of different secondary structures of proteins

(a) -helix (note that it has 3.6 amino acids per turn), (b) -pleated,

and (c) random coil configuration.

The -helical structure arises due to resonance in the peptide linkage

and hydrogen bonding between - NH and >C=O groups along the protein

chains (Fig. 5.10).

Fig. 5.10: Hydrogen bonding between - NH and - C=O groups

250

The hydrogen bonds lie along the axis of the coil, and serve to

maintain the spacing between the turns. This smaller helix in turn is a part of

a larger helix; each turn of a larger helix has nearly thirteen turn of the

smaller helix.

The configuration (secondary structure) of globular proteins is still not

clear. However, it is believed that the globular proteins usually have helical

as well as nonhelical parts in their structures. Most of the globular proteins

such as silk fibroin have their, adjacent peptide chains running in opposite

direction.

(c) Ternary Structure

The tertiary structure is the way in which helical and random coil

regions fold with respect to each other (Fig. 5.11). That is, it refers to the

three-dimensional relationship of amino acid segments that may be far apart

from each other in the linear sequence.

The tertiary structure i.e. the three dimensional structure describes the

overall spatial arrangement of the polypeptide chain (or chains) and

thus give an exact account of molecular shape in most of the small and

medium-sized proteins. The shape of protein is best determined by X-ray

studies. The detailed determination of this shape has carried out only for a

few proteins. In soluble proteins, the overall shape is found to be globular, as

a compact sphere. The shape or a proteins best determined by X-ray studies

which is achieved in many years and only by experts. The three-dimensional

structures of several enzymes are known, the three important are -

chymotrypsin (heaving three chains), lysozome in 1965 (having one chain of

129 residues) and ribnuclease in 1967 (having one chain of 124 residues).

251

At normal pH and temperature, each protein will take a shape that is

energetically most stable. This shape is specific to a given amino acid

sequence and is called the native state of the protein. In general, globular

proteins are very tightly folded into a compact spherical form. This folding

results from interactions between the various side chain groups of

constituent amino acids and may involve several types of forces; hydrogen

bonding, disulphide bridge, ionic or salt bridges and hydrophobic

interactions.

Fig. 5.11: Diagram representing the tertiary and quaternary structure of

hemoglobin. This protein is composed of four subunits; two and . The

sites, in which the four heme groups are located, as well as the amino (N) and

carboxyl (C) termini of the polypeptide chains are indicated.

The highly foaled structure of the protein can be unfolded (opened up)

by means of heat, light, ultraviolet light and by chemical, such as acids,

alkalis, organic solvents, alcohols, acetone, urea and synthetic detergents.

This unfolding process is called denaturation and it might be reversible (e.g.

when the protein is gently treated with urea) or irreversible (when the

protein is violently treated). During denaturation the secondary and tertiary

structures of the protein are completely lost without any break in the primary

252

structure. The denatured protein alters completely in physical properties and

becomes much less soluble at its isoelectric point. Moreover, if the protein

has some biological activity, it is lost during denaturation, e.g. enzymes are

deactivated at elevated temperatures.

(d) Quaternary Structure

Most of the large and even some medium-sized proteins exist as an

association of several chains. In such cases, this protein is known as

oligomeric and the individual chain as promoters or subunits. The

association of sub-units can be disrupted by reagents which do not break

covalent bonds and thus the two chains joined by disulphide bonds should

not be considered as two subunits. Each sub-unit has its own primary,

secondary and tertiary structures, and two or more sub-units in a given may

have identical or different above three structures.

The quaternary structure is maintained by globular and fibrous

proteins. For eq. Hemoglobin is a global proteins & Keratin is, brous

protein, and both of them exhibit quaternary structure.

The quaternary structure is the arrangement of protein subunits within

complex proteins made-up of two or more such subunits. For example, the

hemoglobin molecule is composed of four polypeptide chains, two

designated and two (Fig. 5.11). The chain fit together in an

approximately tetrahedral arrangement in the quaternary structure of

hemoglobin. Separation and association of the subunits may occur

spontaneously. Hemoglobin may be broken into two half-molecules (two

and two ) by urea. When urea is removed they reassemble, forming

complete functional molecules.

253

Check Your Progress - 1

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) The term 'polymer' meaning......................of many............

natural polymers are often called............

(ii) In polysaccharides, the monomer hexose unit linked together

forming............linkage, while in proteins amino acid molecules

link forming........link with the elimination of water molecule.

(iii)The polymerization process generally takes place through.........,

precisely through the.....................condensation of the reactant

molecules.

(b) (i) '' the number of....................in a...............is called the.............

(ii) The secondary structure is the......................of amino acids that are

close to each other in the....................

(iii)The tertiary structure is the way in which and..................... region

fold with respect to.................

(iv)The primary structure of protein is the.....................of................,

while the secondary structure arise due to the regular.............of

the...............of chain due to inter molecular ..................... between

the............... and .................. groups.

5.5 BIOPOLYMER INTERACTION

Properties of biopolymer depend upon intermolecular forces like van

der Walls forces and hydrogen bonds existing in the macromolecules.

Although these intermolecular forces are present in simple molecules also,

254

their effect is less significant in them as compared to that in

macromolecules. This is due to the accumulative effect of these forces all

along the long chains of the polymers. Apparently, longer the chain, more

intense is the effect of intermolecular forces.

Several different-types of bonds are involved in maintaining the four

levels of protein structure. The covalent bond in proteins are of two main

types. The first is the peptide bond uniting amino acid monomers in the

primary sequence. The second is the disulfide bond (S-S bridge) which, as

we have just seen, is established between the -SH groups of two cysteine

residues and is responsible for some aspects of secondary and tertiary

structure.

5.5.1 forces involved in biopolymer interaction

Various kinds of weak interactions are important in the establishment

of secondary and tertiary structure. These weak bonds are all non-covalent;

the main types are as follows:

Ionic or electrostatic bonds result from the attractive force between

ionized groups having opposite charges.

Hydrogen bonds result when a H+ (proton) is shared between two

neighboring electronegative atoms. The H+ can be shared between nitrogen

or oxygen atoms that are close to each other. Hydrogen bonds have many

important biochemical functions. They are essential for the specific pairing

between nucleic acid bases, thus providing the main force that holds the two

DNA strands together as well as allowing the specific copying of DNA into

RNA.

255

Hydrogen bonds in DNA and protein play important part. Thus the

forces that stabilise biopolymer structures are as follows:

(a) Hydrogen bonding: Weak force of attraction between a partially

positive hydrogen and a partially negative atom such as oxygen,

fluorine or nitrogen on the same or another molecule.

(b) Ionic bonding: Side chain cross-linking can occur as a result of

bonding between anionic and cationic side chains.

(c) Covalent bonding: The most common form of inter-chain bonding is

the disulfide bond formed between the sulfur atoms of two cysteine

residues. The insulin consists of two polypeptide chains linked

together by this type of bridges.

(d) Hydrophobic bonding: Several amino acid residues have

hydrophobic (water-hating) side chains. Proteins in aqueous solutions

fold so that most of the hydrophobic chains become clustered inside

the folds. The polar side chains which are hydrophilic (water-loving)

lie on the outside or the surface of the protein.

5.5.2 Electrostatic charges and molecular expansion of hydrophobic

forces

Hydrophobic interactions involve the clustering of molecular groups,

which associate with each other in such a way that they are not in contact

with water. In globular proteins the side chains of the most hydrophobic

amino acid tend to aggregate inside the molecule, and the hydrophilic groups

protrude from the surface of the structure. The hydrophobic residues tend to

256

rapel the water molecules that surround the protein, thereby causing the

globular structure to be more compact.

Van der Waals interactions occur only when two atoms come very

close together. The closeness of two molecules can induce charge

fluctuations, which may produce mutual attraction at very short range.

The essential difference between a covalent and a non-covalent bond

in the amount of energy needed to break the bond. For example, breaking a

hydrogen bond requires only 4.5 kcal mole-1

, as compared with 110 kcal

mole-1

, for the covalent O-H bond present in water. Although each

individual bond is weak, larger numbers of them can produce stable

structures, as in the case of double-stranded DNA. Covalent bonds are

generally broken by the intervention of enzymes whereas non-covalent

bonds are easily dissociated by physicochemical forces.

5.5.3 Dispersion force interaction

Many cellular proteins exist as complexes of multiple subunits that are

held together by weak interactions. For analytical purposes, cell biologists

sometimes find it desirable to dissociate them into their component

polypeptides. Electrical charges of proteins and the iso-electric point.

In addition to the terminal -NH3+ and -COO

+ charged groups, proteins

contain dicarboxylie - and diamino-amino acids, which dissociate as

follows:

1. The acidic groups lose protons and become negatively charged. For

example, in aspartic and glutamic acids, the free carboxyl group

dissociates into -COO + H+.

257

2. The basic groups, by gaining protons, become positively charged:

(-NH2 + H+ - NH3

+)

This is found in amino acids with two basic groups, such as lysine or

arginine.

The actual charges of a protein molecule is the result of the sum of all

single charges. Because dissociation of the different acidic and basic groups

takes place at different hydrogen ion concentrations of the total charge of the

molecule. pH greatly influences the total charges of the molecule shows that

in an acid medium, amino groups capture hydrogen ions and react as bases (-

NH2 + H+ -NH3

+), in alkaline medium the reverse takes place and

carboxylic groups dissociate (-COOH COO- + H

+). For every protein

there is a definite pH at which the sum of positive and negative charges is

zero. This pH is called the isoelectric point (pI). At the isoelectric point,

proteins placed in an electrical field do not migrate to either of the poles,

whereas as a lower pH they migrate to the negative pole (cathode) and at a

higher pH, to the positive pole (anode). This migration is called

electrophoresis, and it provides a useful technique for the separation of

cellular proteins.

5.5.4 Multiple Equilibria and various types of binding process in

Biological system

As has been pointed out earlier, the multiple equilibria involving

condensation of hexose units in polysacharides involve formation of /

glycosidic link (fig. 5.4)with elimination of water. The reverse process,

hydrolysis takes place in presence of an acids or an enzyme. Thus starch

when boiled with dilute acids is hydrolyses completely into glucose.

258

However in presence of diastase enzyme it yields maltose, which in presence

of maltase enzyme gives glucose. Similarly, cellulose when boiled with

dilute H2SO4 is completely hydrolysed in to glucose.

Proteins and polypeptides are condensation products of various amino

acids, which involve multiple equilibria for formation of peptide bonds, with

elimination of water (fig. 5.8) Proteins and polypeptides, on hydrolysis

liberate component amino acids. The hydrolysis process takes place in

different steps, with formation of various intermediate products such as

protease, peptone, peptide:

Protein Protease Peptone Peptide Amino acids

Similarly nucleic acids (DNA, RNA) are condensation polymers

involving three components bases (pyrimidine or purine bases), pentose

sugar (ribose or 2- deoxyribose) and phophoric acid (fig. 5.6), these three

components are regenerated, when these polymeric acids are hydrolysed.

All these biopolymers and their derivatives involve various binding

processes (binding forces) mentioned earlier, viz hydrozen bonding, ionic

bonding, covalent bonding, hydrophobic bonding etc.

5.5 HYDROGEN ION TITRATION CURAVES

In solution the dissociation of each ionizable group in the molecule

may be represented as follows:

COOH COO- + H

+

NH3 NH2 + H+

The dissociated and undissociated forms of each group exist in

equilibrium with each other and the position of the equilibrium (or the

259

tendency of each of the groups to dissociate) may be expressed in terms of

the equilibrium dissociation constant) K, often termed Ka because it refers to

the dissociation of the groups that liberate protons, i.e., acids. The actual

values for Ka are often very small and are conventionally expressed as the

negative logarithm of the value, a term known as the pKa value.

pKa = - log Ka

This function results in a numerical value which is less cumbersome

to use and is comparable with the method of expressing the hydrogen ion

concentration of a solution, the pH value.

pH - - log [H+]

The concentration of hydrogen ions liberated by the dissociation of an

acid is related to the dissociation constant for that acid and this relationship

can be expressed by the Henderson-Hasselbach equation:

acid

saltpKpH a log

Where the square backets indicate the molar concentration of the

named substance.

An examination of this equation reveals the fact that when the

concentrations of salt & undissociated acid are equal, then the pH of the

solution is numerically equal to the pKa of that acid. The lower the value of

pKa of an acid, the greatest is the ability of the acid to dissociate, yielding

hydrogen ions, a characteristic known as the strength of the acid. Amino

acids with 2 ionizable groups, an -carboxyl and -amino group, will be

260

characterized by a pKa value for each group and the actual value will give an

indication of the strength of the acidic or basic group concerned.

The ionization of an amino acid is most easily demonstrated in a

titration curve, which can be prepared by titrating a solution of the amino

acid in the fully protonated form with a solution of NaOH and plotting the

amount of alkali added against the resulting pH of the solution. The titration

curves for a simple amino acid will show two regions where the addition of

alkali results in only a small change in the pH value of the-mixture. The

buffering action of an amino acid is most significant over these pH ranges.

The first end point in such titration is due to the -COOH group and the

pKa value for this is called pKa, while the second pk value is for the amino

group and is called pKa2 In practice each acid and its salt will act as a

buffer over a pH range of approximately one unit on either side of its pKa

value (Fig. 5.12).

For the amino acid alanine where pKa2 is 2.4 and pKa is 9.6, the most

effective buffering action occurs over the pH ranges 2.4 1.0 & 9.6 1.0. In

addition to the -amino and -carboxyl groups, those amino acids with an

extra ionizable group will also have a pKa value. Glutamic acid is an

example of an amino acid with an extra acidic group on the -carbon and

lysine is an example of an amino acid with an extra amino group on the

carbon atom. As a result they each have three ionizable groups and three pKa

values can be demonstrated. The PKa, value being for the extra group other

functional groups present in an amino group acid may also be ionizable and

will hence characteristic pKa values such amino acids results in the complex

titration curves.

261

The overall charge carried by an amino acid depends upon the pH of

the solution and the pKa values of the ionizable groups present, a proton will

be greater than the pKa value for a group, a proton will be last and the

molecule will carry a negative charge but if the pH is less than the pKa,

value, a positive charge will predominate. The fact that at different pH

values different amino acids v/ill be present in different ionic forms and will

carry different net charges is utilized in many analytical method, e.g.

electrophoresis and ion exchange chromatography.

The iso-ionic point of a molecule is the pH at which the number of

negative charges due to proton gain and the zwitter ionic form predominates.

The iso-electric point (pi) is the pH of the solution at which the molecules

show no migration in an electric field and can be determined experimentally

by electrophoresis for, amino acids it is equal to the iso-ionic point. The iso-

ionic point if an amino acid with one -COOH and one -NH2 group is the

mean of the two pKa values. However, when 3 ionizable groups are present,

the effect of an extra acid group will be to reduce the ionic character of the

other acid group and hence the pH value will not be the mean of the 2

separate pKa values but will more closely approximate to the mean of the

closet pKa values.

262

Fig. 5.12

Check Your Progress - 2

Notes : (1) Write your answer in the space given below.

(2) Compare your answer with those given at the end of the unit.

(a) (i) Properties of biopolymers depend upon intermolecular forces like

........... and..................... existing in the macromolecules.

(ii) The forces that stabilize biopolymer structures are

(a) .........................................

(b) .........................................

(c) .........................................

(d) .........................................

(iii) The hexose units in polysaccharides link together using .......... while,

in proteins various amino acids are linked together by...............

(b) (i) The hydrophobic interactions involve the clustering of ............, which

associate with each other in such a way that they are ............

(ii) Nucleic acids are condensation polymers involving three

components:

(a) ................ (................or................)

263

(b) ................ (..............or.............) and (c) .....................

(iii) The Henderson Hasselbach equation is : ..............................

5.6 LET US SUM UP

After going through this unit you would have achieved the objectives

given at the start of this unit. Let us recall what we have discussed so for:

The term polymer is a Greek word meaning combination of many

molecules ('Poly' means many and 'mer' means part molecules or

unit).

Polymers which are obtained only from one type of monomers are

called homopolymers, but those which are obtained by the

polymerisation of two different type of monomers are known as

copolymers or mixed polymers.

Polymers are generally classified into natural polymers and synthetic

polymers. Natural polymers are often called biopolymers. They

include starch, cellulose, proteins, nucleic acid etc.

Most of the polymers are linear in which monomer units are linked

together linearly and form long straight chains. However some

branched chain and three dimension network polymers are also found.

Polymerisation process generally takes place by condensation of the

component units with elimination of water (or HCl, NH3).In

polysaccherides the hexose units are interlinked forming glycosidic

264

link, while in proteins component amino acid (at least 20 type)

molecule condense forming peptide link (-CONH-) similarly nucleic

acids are linear polymers of three component units, a base (purine or

pyrimidine), pentose sugar (ribase or 2-deoxyribose) and phosphoric

acid.

The molecular size of a polymer can be determined by degree of

polymerisation, radius of gyration, hydrodynamic volume etc.

The degree of polymerisation,n, is the number of repeating units in a

macromolecule, and can be obtained by dividing the molecular weight

of the macromolecule by the molecular weight of the monomer.

The root mean square distance gives the dimension of the molecule.

For determination of structure of a polypeptide or a protein it is

necessary to find out (1) how many amino acids are present in the

polypeptide or amino acid chain, and (2) what is the order of linking

of amino acids in the chain.

The analysis of the given polypeptide (or the protein) it is hydrolyzed

with 6N HCl. The amino acids present in the chain are thus separate

apart. Now, isolation and identification of structure of each of the

amino acid is done using the following techniques:

(a) Ion exchange chromatography.

(b) pH dependent precipitation, and

(c) Electrophoresis.

265

After knowing the number of amino acids, the sequence of amino

acids residues along, the peptide chain is determined, using 'End

Group Analysis'. In 'end-group analysis', analysis of the amino

terminal and the carboxylic terminal is done one by one, using various

well known standard methods e.g. Sanger's, Adman's. Densil's and

enzymic methods for amino end degradation and Schlack and

Kempf's, reduction, hydrazinolysis and enzymic methods for carboxyl

end-degradation.

Structure of proteins involves four types of structures. While,

primary structure of protein refers to the number, nature and

sequence of amino acid residues along the polypeptide chain. The

secondary structure is the spatial arrangement of amino acids that

are close to each other in the peptide chain. The -helical structure is

characteristic of proteins and arises due to resonance in the peptide

linkage and hydrogen-bonding between- NH and C=0 groups along

protein chain.

The tertiary structure is the way in which helical and random coil

regions fold with respect to each other. It refers to the three

dimensional relationship of amino acid segments that many be far

apart from each other in the linear sequence.

The quaternary structure is the arrangement of protein submits within

complex proteins made up of two or more such subunits. It is

maintained by globular and fibrous proteins, e.g. haemoglobin and

keratin.

266

Out of the various forces involve in biopolymers hydrophobic

interactions involve the clustering of non-polar groups, which

associate with each other in such a way that they are not in contact

with water.

In solution the dissociation of each ionizable group is the molecule

may be represented as follows:

COOH COO- + H

+

NH3+ NH2 + H

+

The dissociation constant Ka of the equilibria are generally

expressed in terms of PKa which is the negative logarithm of Ka:

pKa = - logKa

The numerical value of PKa is equal to the pH at half

neutralization point, in pH, titration curve, according to the

dHenderson-Hasselbach equation:

Acid

SaltlogpKpH

a

5.7 CHECK YOUR PROGRESS: THE KEY

1(a)(i) combination

molecules

biopolymers

(ii) glycosidic

peptide

267

(iii) condensation

stepwise intermolecular

(b)(i) repeating units

macro molecule

degree of polymerisation

(ii) spatial arrangement

peptide chain

(iii) helical

random coil

each other

(iv) sequence

amino acids

folding

backbone

polypeptide

hydrogen bonding

carboxyl

amino

2(a)(i) wander waals forces

hydrogen bonds

(ii) (a) Hydrogen-bonding

(b) Ionic-bonding

(c) Covalent-bonding

268

(d) Hydrophobic-bonding

(iii) glycosidic linkage

peptide bond

(b)(i) non-polar groups

not in contact with water

(ii) (a) bases (pyramidine or purine)

(b) pentose sugar (ribose or 2-deoxyribose)

(c) phosphoric acid

(iii) Acid

SaltpKpH a log

269

UNIT-6 BIOPOLYMER CHARACTERISATION

Structures

6.1 Introduction

6.2 Objectives

6.3 Thermodynamics of Biopolymer Solutions.

6.3.1 Osmotic Pressure.

6.3.2 Membrane Equilibrium.

6.3.3 Muscular Contraction and Energy Generation in

Machnochemical System.

6.4 Molecular weight Determination of Biopolymers.

6.4.1 Evolution of size, shape and Molecular weight.

6.4.2 Extent of hydration.

6.4.3 Sedimentation Equilibriam.

6.5 Hydrodynamic Methods.

6.5.1 Diffusion.

6.5.2 Sedimentation Velocity.

6.5.3 Viscosity.

6.5.4 Electrophoresis and Rotational Motions.

6.6 Let Us Sum Up

6.7 Check Your Progress: The Key

270

6.1 INTRODUCTION

Biopolymer characterization is a must for any worker dealing with

these. The information is obtained for the selection of polymers for specific

properties. Special techniques have to be employed for composites and

polymer blends, viz. precipitation, fractional extraction, turbidinistry and get

permeation chromatograph.

NMR provides both in solid state as well as in conventional solution

information on molecular motion, chain flexibility, crystatllinity and

configuration due to chain entanglement or cross linking.

Infrared spectroscopy and Raman spectroscopy using laser beam

provide information on chemical, structural and conformational aspect of

polymers and polymer blends, stress induced charges and chemical

reactions.

Pyrolysis Gas Chromatography (py-ge):

In this technique the polymers are converted to lower molecular

weight products by the action of heat. This technique is extensively applied

to polymers. It provides quantitative analysis of polymeric structure,

monomer composition stereochemistry, tacticity and monomer arrangement

in homo and copolymers.

Mass Spectrometer:

This technique is most useful when combined with pyrolysis or py-ge.

Alone it is applied to molecular weight up to 5000 or less.

271

UV spectroscopy is not suitable but sometimes it is used in the

determination of additives, stabilizers and other minor impurities.

Luminescence Spectroscopy:

This technique provides useful information on polymers through the

emitted light. This technique can be applied to powders, film, fiber or

solution. This is used in studying crystallinity, internal molecular motion,

and changes in molecular conformation. The polymer is sometimes doped

with a fluorescent dye.

X-ray photoelectron spectroscopy (Xp) is a useful technique for

structural and chemical characterization of polymer surfaces energies. The

sample is exposed to monoergetic beam of soft x-rays and the kinetics of the

electrons photoemitted surface are measured. Previously it was used for

fluoropolymers but recently they are used to study adhesion, weathering

phenomenon, degradation of surfaces and diffusion of additives.

Polymer chain interaction may result in an ordering of chain in

crystalline or amorphous phases. The small changes in size and distribution

of these ordered region may bring about differences in physical and other

properties in different samples.

The forces responsible for these are Vander Waals, dipole-dipole or

ionic in nature and are known as secondary bonding. In some polymers

crystalline and amorphous regions are usually bridged by polymer chains

extending from one phase to another.

272

Tm and Tg are factors that guide the degree of chain motion. Below Tg,

the polymer is usually brittle and glassy. The semi-crystalline polymers

exhibit best mechanical and physical properties, between Tg and Tm. Above

Tm the polymer flows under stress. The geometry, size, and distribution of

crystalline region usually depend on the thermal and mechanical history of

the polymer.

Characterization of Molecular Order:

Various methods employed for this are thermal methods. X-ray

diffraction, solid state NMR, infra-red Raman spectroscopy, microscopy,

inverse gas chromatograph, reaction scattering, etc.

It is possible for the polymers to have the average molecular weight

but different molecular weight distribution (MWD). Molecular weight

distribution has been found to be an important variable and contributes to

physical properties. Size exclusion chromatography is used for the study of

molecular weight distribution.

During the process of synthesis of polymer, the growth of a polymer

chain depends upon the availability of the monomers in its vicinity which

differs from one place to another in the reacting mixture. As a result, a

polymer sample contains chains of a varying lengths and, therefore, its

molecular mass is always expressed as an average. In contrast, natural

polymers such a proteins, contain chains of identical length and hence their

molecular masses are singular in nature.

Statistically, we can express the average in terms of number or

weight. Consequently, the molecular mass of a polymer is expressed as

number average molecular mass (M n ) or weight average molecular mass

273

(M w ). The ratio of the eweight and number average molecular mass

(M w /M n ) is called poly dispersity index (PDI). In natural polymers, which

are generally monodispersed, the PDI is unity (i.e. M w =Mn ).

In synthetic polymers which are always polydispresed, the PDI is

greater than unity, because M w is always higher than M n .

M n is determined by employing methods which depend upon the

number of molecules present in the polymer sample, viz. colligative

properties like osmotic pressure, depression in freezing point and elevation

in boiling points. On the other hand, methods such as light scattering and

ultra-centrifuge depend on the mass of the individual molecules and yield

weight-average molecular mass.

6.2 OBJECTIVES

The main aim of this unit is to discuss characterization and

determination of molecular weight of biopolymers. After going through this

unit you would be able to:

describe various methods of characterization of biopolymers,

discuss thermodynamics of biopolymer solutions,

understand importance of osmotic pressure and its determination, and

explain methods of molecular weight determination of biopolymers.

6.3 THERMODYNAMICS OF BIOPOLYMER SOLUTIONS

The solution of a polymer is a function of molecular structure,

composition, and molecular weight.

274

Polar polymers are usually more soluble in polar solvents, e.g.,

polyvinyl alcohol, in water, where as non-polar polymers are more soluble in

non-polar solvents, e.g., polystyrene in toluene.

In crystalline polymer the inter-molecular crystalline forces must be

overcome by the solvent. Cross -linked polymers swell in a compatible

solvent rather than dissolve. The rate of solution decreases with increasing

molecular weight and increasing length of side chain launching.

lnlnln sososo STHG

The dissolution of a polymer is favored if H < TDS.

Thus for solubility to occur H must be negative or if positive must

be very close to zero.

2

2121 )( EH

where F is the change in internal energy on forming a solution, 21 and are

volume fractions of polymer and solvent and 21

and are solubility

parameters of the polymer and the solvent. Thus for solubility 5.0)( 21

for solubility.

2/1)( npd

where d ,is due to dispersion or Vander Waals forces, p , is permanent

dipole interaction and n

is due to hydrogen bonds.

This is more helpful where two non-solvents can form a solution

mixture. This approach is important in the coating fields.

275

Thus the viscosity of a polymer solution or the stability of a

suspension of polymer particles are affected by the solvent. Crystallization

and morphological changes are induced by solvent.

Various solution methods are used for determination of molecular

weights of macromolecules.

6.3.1 Osmotic Pressure

The properties of solution that depend on the number of particles

without any consideration of their kind, are called colligative properties

(Colligates-collected together). Thus most of the thermodynamicall

properties like lowering of vapour pressure, elevation of bonding point,

depression of freezing point, osmotic pressure etc., come under colligative

properties. Various colligative properties are used for determining molecular

weight of substances in solution particularly osmotic pressure.

Osmotic is defined as the process of net diffusion of water molecules

from a dilute solution or pure water (solvent) itself to a more concentrated

solution, when both are separated by a semipermeable membrane.

This membrane allows the water to diffuse but not the solute. Thus,

separated from water by semipermeable the membrane. Water molecules

diffuse in both directions across the semipermeable membrane, but a net

diffusion or osmosis of water from the dilute to the concentrated solution

results from a larger number of water molecules diffusing in that direction

than in the reverse direction. Water continues to flow into the more

concentrated solution across the membrane in this way until the hydrostatic

pressure rises so high on the concentrated side of the membrane to cause a

transmembrane diffusion of water in the opposite direction at the rate as the

276

osmotic inflow. This hydrostatic pressure which exactly balance the osmotic

influx of water from pure water to concentrated solution is called the

osmotic pressure of that solution. Thus, osmotic pressure (g) can also be

defined as the pressure which has to be exerted on the concentrated solution

which has to be exerted on the concentrated solution, separated from pure

water by a semipemeable membrane. In order to counteract and stop the

osmotic inflow into the solution. It equals the difference between the

hydrostatic pressures on the two sides of membrane. Osmotic pressure is a

colligative property of a solution. A rise in the number of solute particles in

the solution increases the number of solvent particles bound by solute

particles in complexes of solvent particles bound by solute particles in

complexes called solvates. Osmotic pressure may be considered to be caused

by the higher partial pressure of solvent molecules on that side of the

semipermeable membrane as has higher concentration of the solvent.

Osmotic pressure of a dilute solution is directly proportional to

other colligative properties like this fall in freezing point, lowering

of vapour pressure, etc.

Osmotic pressure is inversely proportional to the MW of the solute.

Colloid osmotic pressure of plasma proteins partly counteracts the

filtering effect of blood pressure and retains water in the plasma. In

Kwashorkor, hepatic cinnboses and nephrosia a fall in plasma concentration

of albumin, refuses the colloid osmotic pressure of blood and lowers the

relation of water in circulation leading to oedema.

6.3.2 Membrane Equilibrium

277

It was Pfeiffer who first deposited cupric ferrocyanide on the pores of

earthenware pot and used it as a semipermeable membrane. Since then a

large number of substances ranging from animal bladder to cellulose have

been used as semipermeable membrane. The semipermeable membrane is

critically important in osmometry. The reliability of osmotic measurements

depends to a considerable extent on proper choice of' membranes. Selection

of a membrane involves reconciliation of high permeability towards the

solvent with virtual impermeability to the smallest solute molecules present

in the solution. The membrane should not swell to any great extent in the

solvent and it should have sufficient fine pores to allow the solvent

molecules to pass through freely.

The most convenient material used is cellulose in the form of a non-

water proofed cellophane sheet or specially treated films of denitrated

cellulose nitrate. Generally cellophane membrane is used because treatment

of cellophane with ammonia solution or other reagents increases the pore

size of the membrane very little. It is possible to prepare membranes of

varying porosity depending on the swelling and solvent transfer treatment.

Hookway

has reported some fast membranes permeable to solutes of

molecular weight 50000, while nitrocellulose membrane can be used up to

molecular weights of 2000. To condition the membrane in water for use with

organic solvents, it is essential at first to wash it with 25, 50, 75 and 100 per

cent alcohol or acetone solutions and then displace the alcohol or acetone by

similar washings with the desired organic solvent. Precaution should be

taken that the membranes is not allowed to dry out. It should always be

stored in the solvent.

278

Theories of Semipermeable Membranes: There are three theories of

sernipermable membranes, viz. sieve theory, solution theory and adsorption

theory.

Sieve theory Traube considered the semipermeable membranes as atomic or

molecular sieves or bundles of capillaries through which larger molecules

find it difficult to diffuse. According to him, the only difference between

various membranes (copper ferrocyanide, parchment, etc.) is in the size of

their pores. This theory fails to explain as to why a rubber membrane is

impermeable to water but permeable to a large molecule like benzene and

pyridine.

Solution theory: Liebig and Hermite postulated that a membrane will be

permeable to substances that dissolve in it and impermeable to those that do

not dissolve which explains why rubber is permeable to benzene, toluene,

pyridine, etc., which are soluble in rubber and diffuse through it, but water

which is insoluble does not pass through it. It can be concluded from the

above that substances that diffuse through the membrane, first dissolve in

the membrane. Although this proved to be a necessary criterion, but it was

not all. Bigelow and Bartell observed osmotic effects with inert substance

where neither solution nor chemical reaction was possible: Porus cups, with

very fine pores or pores clogged with substances, acted as semipermeable

membranes. Silica, carbon, metallic copper, silver, gold can be compressed

into disks with very fine pores also acted as semi-permeable membrane.

Bartell concluded that copper limit of the pores should be 9.0 x 10-3

cm.

The adsorption theory: Wieser and other workers have shown that inert

membranes sometimes take up relatively more of the solvent than the solute.

279

This is known as negative adsorption and the solution gets more

concentrated. Mathieu observed similar phenomenon with a number of

solutions using porus plates as membranes. He concluded that with

sufficiently fine capillaries only water would be adsorbed. Similarly sugar

was negatively adsorbed by copper ferrocyanide membrane. Thin palladium

foil is permeable to hydrogen but impermeable to nitrogen.

Thus a semipemeable membrane acts like a solvent than like a sieve.

Irrespective of the mechanism by which the semipermeable membrane

operates, the chemical potential of the diffusing component is the same on

both sides, of the membrane.

Methods of Measuring Osmotic Pressure: There are three methods for

measuring the osmotic pressure viz. static method, dynamic method and half

sum method.

Static Method: In the static method, the solvent is allowed to diffuse

through the membrane until there is no further interchange in the internal

head h. The osmometer measures the equilibrium difference of level.

Corrections for the effect of surface tension and the resultant equilibrium

concentration of the solution due to passage of solvent through the

membrane has to be applied. Sometimes during the establishment of

equilibrium, which usually is a long period, the concentration of the solution

near the membrane may increase due to adsorption of solvent. This can be

avoided by the proper production and storage of the membrane in the

solvent. This is a simple method but takes unusually long period for the

attainment of the equilibrium.

280

Dynamic Method: In the dynamic method, the solvent flows through the

membrane. The rate of penetration is measured by applying an external gas

pressure to the solution. The interpolated pressure for zero rate will be equal

to osmotic pressure. By establishing an equilibrium pressure which remains

constant for quite a good period give more reliable results. This is a quick

method but requires a leak tight complex cell.

Half Sum Method: The internal head h is adjusted initially to be close to

the expected equilibrium, say slightly above the equilibrium value. Frequent

readings of the depression in volume with time are taken. After sufficient

time the cure between h and time is plotted as represented by x. The

experiment is repeated by adjusting the head slightly below the equilibrium

value and the increase in volume with time is noted till the g curve

represented by Y becomes asymptotic. By calculating the one half the sum

of the ordinates of x & y at several values, a new curve A is obtained which

converges to a constant value. Since in both the cases, the change in volume

involved is small, the equilibrium concentration is assumed equal to the

critical concentration.

6.3.3 Muscular Contraction and Energy Generation in

Machnochemical System

Transmission of a nerve impulse from the nerve to a muscle across the

neuromuscular junction is called neuromuscular transmission. This is

achieved by the release of acetylcholine which acts as neurohumer

transmitter. Calcium helps in the release of acetylcholine form the storing

vesicles present in the nerve ending.

281

Calcium ions neutralize the negative charge of myosin which then

combines with negatively charged action and thus the contraction takes

place. Calcium ions (Ca++

) also activate the myosin ATPase which in term

breaks down the ATP to supply energy required for muscular contraction.

Further the relative amounts of the energy-producing nutrients in the

diet vary widely on the geographic environment and the economic status of

the individual for example, some diet may consists of as much as 80%

carbohydrates, while other diets, such as the diet of Eskimo, may contain

only protein and fats. This large variation in energy producing nutrients has

no effect on the energy requirements of the individual, since the major

metabolic processes of all the three energy producing nutrients (the

biomolecules: protein, carbohydrate and fats) are interrelated through the

Kreb's cycle as indicate in fig. 6.1

Fig. 6.1: Interrelationship between the metabolic processes of the energy-

producing nutrients

MECHANISM OF MUSCLE CONTRACTION

Unfortunately, the precise chemical and physical events that take

place during muscle contraction are yet not fully understood. However, the

Fig. 6.2 illustrates the commonly suggested method of skeletal muscle

282

contraction which shows a relaxed state of the myofibril in Fig. 6.2(a) and a

contracted state in the Fig. 6.2 (b).

It will be noted from the figure that neither the actin nor the myosin

filaments shorten during contraction, rather actin filaments simply slide like

pistons inward among the myosin filaments so that the opposite ends of the

actin filaments (usually barely overlapping in the resting i.e. elongated state

of muscle) from two sides overlap one another considerably while the two

Z-membranes to which actin filaments of two sides are attached approach

the ends of myosin filaments and in this way the length of sacromere is

decreased as a whole. Since, the entire length of the myofibril is composed

of such sarcomeres, the entire myofibril contracts in length and this results in

the contraction of the whole muscle fibre.

Fig. 6.2: The relaxed and contracted states of a myofibril, showing

sliding of the actin filaments into the channels between the myosin

filaments (see also Fig. 6.3)

Answer of the questions how all this is possible is the electron

microscopic structure of actin and myosin filaments (Fig. 6.3) which shows

that myosin molecules are arregated with their heads pointed in one direction

283

along half of the filament and in the opposite direction along the other half.

The head serves as the cross bridges projecting toward the actin filaments at

very 435Ao along the axis of the myosin.

Fig. 6.3: Schematic illustration of possible mechanism of contact of actin

and myosin filaments resulting in muscular contraction.

Furthermore, since there are six actin filaments arranged hexagonally around

the myosin filament, the cross bridges are repeated six times around the

circumference of the myosin filament, arranged helically along the axis of

the myosin. On the other hand, the actin filament is composed of two long

fibrilar actin molecules wound spirally around each other with a complete

turn at each 700Ao. Also, it has a reactive site along its axis occurring once

every 405Ao. It is believed that these reactive sites in some way interact with

the ends of the cross-bridges to provide the force required for pulling the

actin filaments inwards among the myosin filaments. This requires all the

elements of the force provided by the interaction between cross bridges of

myosin and reactive sites of actin filaments in one of the A bands to be

oriented in the same direction and that the direction of the force be reversed

in the other half. This actually happens and can be explained by the

arrangement of the myosin molecules themselves - pointing in the same

direction in half of each myosin filament and in the opposite direction in the

other half as shown in Fig. 6.3.

284

INITIATION AND STOPPAGE OF MUSCLE

CONTRACTION

When the action potential spreads along the muscle fibre, it causes the

electrical current to flow deep, into the interior of the muscle fibre by way of

the T tubules. When the current reaches the triads, Ca++

are released into the

surrounding sarcoplasm and so the normal; concentration of Ca++

in

sarcoplasm which is only 10-7

molar is increased beyond 2x l0-4

molar-

minimum concentration of Ca++

needed to cause muscular contraction.

Within a ten thousand of a second, these ions diffuse the interior of the

myofibrils where they form actomytosin-Ca++

complex and catalyze an

enzyme reaction associated with myosin, to cause the hydrolysis of ATP

which thus yields the energy required for the contraction of myofibril as

discussed in Ratchet theory. As the electrical current caused by the action

potential is over the longitudinal tubules almost immediately reabsorb the

Ca++

out of the sarcoplasm actively and the fibres subsequently relax.

Meanwhile the ADP is regenerated to ATP. The action potential lasts only 1

millisecond while the muscle twitch may be ten to hundred times as long in

duration. Hence, the contractile system can be reactivated long before the

contraction has begun to sub-side. Hence, the muscle can be effectively

tetanised by appropriate frequent stimuli.

Sequence: Action potential conducted via transverse tubule

(t.t.)depolariffilion of terminal cistern (t.c)liberation of Ca++ activation

of meosin ATP ase (ATP-ase;)liberation of energy from splitting of ATP

attached to cross bridges of myosin filaments. Energy E causes actomyosin

contraction.

285

Theories for muscle contraction: As mentioned in the beginning of the

mechanism of muscle contraction that the exact mechanism of muscle

contraction is yet unknown so various theories and hypotheses have been put

forward by different workers. Important ones of these are discussed below.

The Ratchet theory: Also known as the sliding filament theory was put

forward by Huxley el al. According to this theory it is believed that first in

the resting state because of negative charges of the ATP bound with the

cross bridges of the myosin filaments and the negative charges of the actin

filaments, these filaments remain separated from each other with no

attractive forces between them. However, this state of affairs can exist only

in the absence of the Ca++

. On the appearance of Ca++

the following events

occur:

(1) Ca++

binds with negative reactive sites on the ATP on the myosin

cross bridges, and at the same time with the negative re-active sites on the

actin filaments forming the actomyosin-Ca complex.

(2) Cross bridges having strong negative charge in the resting state

due to presence of ATP, normally project straight outward from the myosin

filaments because the shaft of the filament is also negatively charged. When

Ca++

binds with the ATP on the cross bridges, the negativity of the cross

bridges gets neutralized and therefore, the bridges now bend inward towards

the axis of the myosin filament. This also pulls the actin filament, thus

shortening the muscle

(3) When the cross bridges fold is against the shaft of the myosin

filament, the myosin ATPase (activated due to the presence of Ca++

) activity

of the myosin filament causes the ATP to split immediately to ADP. This

286

breaks the actinomvosin - Ca complex, but in the mean time the actin

filament has already been pulled towards the centre of A bands among the

myosin filaments.

(4) Subsequent similar reactions occur at other cross-bridges and the

actin filaments are pulled another step.

(5) Energy from other sources, such as from high energy creatine

phosphate causes almost immediate reconstitution of the ADP to ATP.

Therefore, the cross-bridges that had folded inward now bend outward again

and bind with other Ca++

to pull the actin filament another step.

Thus, by a series of "ratchet" (bonding and pulling, bonding and

pulling) an action similar to that of a man pulling in a rope hand over hand,

the actin filaments theoretically are pulled inward among the myosin

filaments.

The Electrostatic Solenoid theory: Though the ratchet theory has

been widely accepted, there is one well-known valid criticism against it

which may make it untenable. According to this criticism, as the myofibrils

contract they swell also in diameter so that the filaments spread further apart

and their interdistances may be increased as much as 50Ao. Due to small size

of Ca++

which are supposed to link the actin and myosin filaments, it is

doubtful that these could act through such long distances. Therefore, it

seems more likely that the attractive forces between the actin and myosin

filaments are perhaps electrostatic in nature and able to act through long

distances rather than actual chemical bonds. To explain this, several theories

have been put forward and electrostatic solenoid theory is one of them.

287

According to this theory Ca++

do not form actin myosin-Ca complex

rather they bind with myosin only to make these electro-positive while actin

filaments remain electronegative, and therefore, the two types of filaments

are attracted towards each other by electrostatic forces produced due to

different electric charges on the actin and myosin. If we assume that the

filaments can slide among each other but can't touch each other the

electrostatic forces will be as shown by the diagonal lines in the fig. 6.4.

From the figure it should be clear that at the ends of actin filaments

the forces have vectors that extend linearly along their axes, which will

cause the actin filaments to be pulled inward among the myosin filaments

but when the ends of the actin filaments overlap each other, the linear forces

of attraction between the filaments will then occur mainly at the ends of the

myosin filaments and not at ends of the actin filaments. Therefore, the

strength of contraction should theoretically fall off; this indeed, is known to

occur.

Fig. 6.4: Schematic illustration of forces (the oblique lines) that

theoretically develop between actin and myosin filaments charged

differently from each other, illustrating the so called "electrostatic

solenoid" theory of muscle contractions.

This theory is supported by the following two fact:

(1) A difference in electrical charges on the metallic plates

interdigiated in the manner as shown for actin and myosin, will cause inward

288

movement and can be easily demonstrated.

(2) Calculation of the electrical potential difference between the atcin

and myosin filaments that will be required to cause full strength skeletal

muscle contraction (2 to 3 kg/cm2) show that an electrical potential

difference of only 70 mv., would be sufficient. This is less than the action

potential itself, illustrating that the potential required for the function of the

electrostatic solenoid mechanism is easily attainable.

Energetics of muscular contraction: Immediate source of the

energy required for muscular contraction is ATP provided by oxidative

phosphorylation but not at a sufficient rate to sustain muscle during bursts of

intense activity. Consequently, a store creating phosphate which, by acting

as a source of high energy phosphate for prompt resynthesis of ATP as

shown in the fig. 6.5 serve to maintain adequate amounts of ATP. The

transfer of high energy phosphate from creatine phosphate to ADP (the

Lohmann reaction) is catalysed by the enzyme, creatinekinase. The reaction

is reversible, so that resynthesis of retained phosphate can take place when

ATP later becomes available, as during the recovery period following a

period of muscular contraction. Transfer of high energy phosphate from

ATP to creating to from creatine phosphate is catlysed by another enzyme.

ATP creatine transphosphorylase.

289

Fig. 6.5: Formation and breakdown of creatine phosphate and the

relationship of these events to ATP in muscular contraction.

There is still another source of energy in muscle due to presence of an

enzyme, myokinase (adenylate kinase), which catalyses the transfer of a

high energy phosphate from one molecule of ADP to another to form ATP

and AMP.

ADPATPADP Myokinase 2

Muscular fatigue: It is of common knowledge that sustained

contraction of the muscles or repeated muscular contractions lead to

voluntary fatigue. Presumably this fatigue is due to changes in the muscles

themselves and not due to failure of the action potential contraction

coupling. The changes in the muscles which might be responsible for fatigue

are (i) anoxia and (ii) accumulation of metabolic waste products. Although

both these changes are offset by blood flow, there are mechanical problems

in maintaining blood flow during sustained contraction for the rise in

intramuscular tension tends to prevent the passage of blood.

290

Check Your Progress - 1

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) It is possible for the polymers to have the......................but

different..................................

(ii) The rate of solution..................with increasing and .............. of

side chain launching.

(iii)During osmosis the....................allows the...................

to..............but not...................

(b) (i) Ca++

ions neutralize ..................... of ................. which then

combines with negatively charged ................... and this

the...................takes place.

(ii) Theories of muscle contraction are:

(a) ..................................

(b) ..................................

(iii)Another source of energy in muscle is due to pressure of an

enzyme.............., which catalyses the of a................from one

molecule of ADP to another to form...............and AMP.

6.4 MOLECULAR WEIGHT DETERMINATION OF

BIOPOLYMERS

It was Avogadro who developed the concept of a mole. According to

him a mole consists of amount of substance containing the same number of

291

atoms as are present in 12 g of C12

. This has a value 6.0229x1023

. The

weight of a mole in grams is numerically the same as the weight of a single

molecule in atomic mass units (amu). The weight of one gram atom of an

element is called gram-atomic weight and of one mole of molecules is

referred to as gram molecular weight. They are simplified as atomic and

molecular weight. One amu is equal to 1.66x10-24

g. The molecular weight

is the sum of all the atoms present in the molecule.

Molecular Mass of Polymers

Natural polymers such as proteins contain chains of identical length.

Therefore, their molecular masses are singular in nature. On the other hand,

during the process of synthesis of polymers, the growth of a polymer chain

depends upon the availability of the monomer units in its neighbourhood

which differs from one place to another in the reaction mixture. Hence all

the polymers are heterogeneous with respect to molecular weight. The

values of molecular weight of the same polymer will differ with the solvent

and method of determination. A series of polymeric compounds having the

same chemical structure but differing only in molecular weight is known as

polymeric homologous series. The molecular weight so determined will give

the value of average molecular weight. Depending on the method of

determination, different average molecular weights are obtained. They are

given as follows:

(a) Number average molecular weight n

M

(b) Weight average molecular weight w

M

(c) Viscosity average molecular weight v

M

292

(d) Z-average molecular weight z

M

In the case of mono-dispersed systems:

n

M = w

M = v

M = z

M

while they differ very much in polydisperesed system.

Number Average Molecular Mass (n

M )

It is obtained by dividing the sum of masses of all the molecules of

different monomer units of different masses by the total number of

molecules. We can understand it by considering a polymer made up of three

monomeric units of masses M1, M2, & M3,. If N1 molecules of monomer of

mass M1, N2 molecules of mass M2 and N3 molecules of mass M3 constitute

the polymer then,

the total mass of N1, molecules = N1M1 (i)

the total mass of N2 molecules = N2M2 (ii)

the total mass of N3 molecules = N3M3 (iii)

Adding (i), (ii), and (iii), we will get the total mass.

Total molecular mass = N1M1 + N2M2 + N3M3 (iv)

and total molecules = N1 + N2 + N3 (v)

Number Average Molecular mass=N1M1+N2M2+N3M3 (N1+N2+N3)

Ni

MiNinM

293

Where ............321

NNNNi

Where ............332211

MNMNMNNiMi

nM is generally determined by osmotic pressure measurement,

depression in freezing point and elevation in boiling point.

Weight Average Molecular Mass ( wM )

It is obtained by multiplying the sum of total molecular masses of

different monomeric units by their respective molecular masses, adding all

the molecular masses and then dividing by the total mass of all the

molecules.

If a polymer consists of N1 molecules of monomeric unit of molecular

mass M1,N2 molecules of another monomeric unit of molecular mass M2 and

N3, molecules of the third monomeric unit of molecular mass of M3 then

Weight average molecular

mass = 332211

333222111

MNMNMN

MMNMMNMMN

(vii)

or wM = 332211

2

33

2

22

2

11

MNMNMN

MNMNMN

(viii)

or wM = NiMi

NiMi

2

(ix) Where

2NiMi = 2

33

2

22

2

11MNMNMN ..................

and NiMi = 332211

MNMNMN ..................

294

wM is generally determined by ultracentrifugation or sedimentation

e.g. the number average molecular mass and the weight average molecular

mass of a polymer sample containing 30 molecules of molecular mass

10,000, 30% of molecular mass 20,000 and the remaining 40% of molecular

mass 30,000, will be-

n

M = 403030

000,3040000,2030000,1030

= 000,21100

2100000

100

1200000600000300000

wM = 000,3040000,2030000,1030

000,30000,3040000,20000,2030000,10000,1030

= 1200000600000300000

03600000000012000000003000000000

= 2100000

05100000000=

21

510000= 24286

Ratio of wM is called 'Polydispersion index' (PDI)

PDI = nM

wM

For natural polymers, PDI = 1 whereas for synthetic fibres, PDI>1.

6.4.1 Evalution of size, shape and Molecular weight

As has been pointed out polymer chain interaction may result in an

ordering of chains in crystalline or amorphous phase. The small changes in

295

size and distribution of these ordered regions may bring about differences in

the physical and mechanical properties. It is a difficult task to characterize

cross linked structures. Sometimes solution techniques are used where

uncross link polymers are dissolved. True cross linked polymer networks are

of infinites size with many cross-linked sites per chain forming a three-

dimensional network of great complexity.

The molecular size can be determined by decree of polymerisation,

radius of gyration, hydrodynamic volume etc.

The root mean square end distance gives the dimension of the

molecule. This can be determined by the viscosity or light scattering of

dilute solutions.

The structures of biopolymer, as has been pointed in unit-5, is

determined using N.M.R. studies, ion exchange or electrophoresis

techniques.

Molecular Weight Determination

The molecular weights can be determination by ebiometry, cryscopy,

osmometry, end group determination, light scattering, ultra centrifuge and

dilute viscometry (Table 6.1.)

296

Table 6.1

Molecula

r weight

size of

Symbol Method of

Determining

Molecul

e

Number

average ...332211

iii

n

n

Mn

n

Mn

n

MnM

Colligative

method

small

Weight

average ...

2

33

2

22

2

11

iiiiii

w

Mn

Mn

Mn

Mn

Mn

MnM

Light

scattering

large

Average ...

2

3

33

2

3

22

2

3

11

iiiiii

z

Mn

Mn

Mn

Mn

Mn

MnM

Sedimentatio

n method

large

Viscosity a

vv MKM 1 Viscometer

method

Where n = number of molecules in sample.

n1, n2, n3 = number of molecules of molecular weight M1, M2, M3

M1, M2, M3 = molecular weight of individual molecules

The molecular weights can be determined directly or in solution.

Direct measurement

The method for direct determination of molecular weights can only be

applied to gases or volatile liquids and solids. They are known as:

1. Vapour density method

2. Low pressure effusion of gases

3. Mass spectrometry

297

These methods are useful for gaseous or volatile substances. Hence,

molecular weights of biopolymers are generally determined by solution

methods.

Solution Methods:

There are two types of important methods of determining the

molecular weight of substances in solution, viz. chemical method and

physical method.

Chemical Method:

Chemical methods are important for the molecular weight

determination of macromolecular compounds. Application to this method

requires that the structure of polymer should contain a known number of the

chemically determinable functional groups, radicals or elements per

molecule. In polymers these functional groups occur as end groups ( )

except branched polymers. In linear polymers the quantitative determination

of all end groups, e.g., a polyester where-OH groups is at one end and-

COOH group at the other end, can be estimated by acidimetric titrations.

Therefore determination of one of the functional groups suffices for the

evaluation of number average molecular weight. Katchalski determined the

molecular weight of polyamino acids by the chemical, as well as by other,

methods and found them in reasonable agreements.

In case the polymer is formed by chain transfer the number of transfer

agents molecules in the polymer can be determined by chemical analysis.

From these the molecular weight of the polymer can be computed.

)(

)(

)(

111

M

SC

DPDPonn

298

where DPn is the number average of polymerization

onDP )(

1is the

reciprocal of DPn without chain transfer agent or solvent, and (S) & (M) are

the concentrations of chain transfer agent and monomer respectively and C1

is the chain transfer constant.

p

i

b

s

b

b

m

MK

KCand

K

KC

K

KC

1,

where Km, Ks and Ki are the velocity constants of chain transfer with

monomer, solvent and initiator respectively.

Temperature, 'C'

Fig. 6.6

In the case of branched chain polymer, the end group determination

establishes the branching of chains. Kern19

polymerized styrene in presence

of bromobenzoyl-peroxide and determined the amount of bromine in the

polymer. He found that each molecule contains three to four atoms of

bromine. In another sample polystyrene containing hydroxyl as end groups

and the presence of end groups was ascertained by infrared spectroscopy.

The molecular weight was found to be 17,300.

299

In another method, the presence of unique structural unit in small

amount was the sufficient data for finding the molecular weight, e.g., a

protein molecule may contain just one atom of ion. If Aa is the atomic

weight of this trace element and x is the analytically determined percentage,

then the molecular weight M

M = 100Aa/x

Hemoglobin from mammalian blood contain 0.335 per cent ion

700,16335.0

85.55100

M

Similarly small amounts of amino acids, sulphur, and other specific

constituent can be used to determine the molecular weight of the polymer.

Tablet 6.2 gives the molecular weight determined by chemical

method.

Table 6.2

Molecular weight

Egg Albumin 44,000

Hemoglobin 16,700

Insulin 12,000, 6000

Edestin 46, 000

In the case of copolymerization of two monomers of M1, & M2, the

copolymer will be formed on the basis of their respective reactivities r1 & r2,

21

22

2

12

11

1K

Krand

K

Kr

300

where K11, K12 are the rate constants for the reactions M1, free radical

and monomers M1, M2 and K22, K21 are the rate constant for the reactions M2

free radical with monomer M2, & M1.

Generally 121rr

but sometimes r1 = r2 = 1, then random copolymers will result. If r1 &

r2 are low and r1r2 is approaching zero, alternating copolymers will result.

If edchM

edchMM

oarg

arg

2

1

& 2

1

int

int

Mmonomerialofmole

MmonomerialofmoleP

o

then it can be shown that.

1

2

1

21

2

2

]4)1[()1(

r

rrPPPM

ooo

o

Therefore if the composition Mo is allowed to copolymerize, a

copolymer of desired composition can be obtained and the molecular weight

can be computed.

The chemical methods only find use in condensation polymers which

have average molecular weight of the order of 25,000. This method is not

sensitive to large molecular weights sometimes some of end groups not

considered for computing the molecular weight becomes consequential,

especially in the case of cross-linked and highly branch chained structures.

Physical Methods

301

Various physical methods depend on either the evaluation of

thermodynamic properties or the kinetic behavior or the combination of the

two, in dilute solutions. Polymer solutions exhibit large deviation from their

limiting infinite dilution behavior. Hence, not only, all the experiments are

carried out at low concentrations but they are invariably extrapolated to

infinite dilution. In the case of chain molecules, a polymer molecule is

assumed to have a symmetrical statistical distribution of chain elements

about a centre of gravity and the volume occupied by this distribution is

many times the actual molecular volume. Thus the volume over which an

individual polymer molecule exerts its influence depends on the chain length

and on the interaction between the polymer and the solvent. Hence the

solution taken for physical measurements should be so dilute that each of the

molecule couples separate portion of the volume. This will not only require

very sensitive equipment to measure small physical changes, but the

polymer should also be very properly fractionated, otherwise the molecular

weight obtained by different methods will vary in wide range.

Colligative Properties

The dilute solutions show more or less ideal behavior as the heat and

volume changes, accompanying the mixing of solute and solvent are

negligible for all practical purposes. Dilute solution obey Raoult's law.

Dilute solutions containing non volatile solute exhibit some special

properties which depend only on the number of particles in the solution

irrespective of their nature. These properties are termed as:

Colligative properties, and these include:

(i) Lowering in the vapour pressure

302

(ii) Elevation in the boiling point

(iii) Depression in the freezing point

(iv) Osmotic pressure

The important of these properties lies in the fact that they provide

methods for the determination of molecular weights of dissolve solute.

Lowering in Vapour pressure

When a nonvolatile solute is dissolved in a solvent, its vapour

pressure decreases, Von Babo showed that although both vapour pressure of

pure solvent (po) and vapour pressure of solution (ps) increase with increase

of temperature yet the ration, o

so

p

pp remains the same at all temperature.

While po-ps is the lowering in vapour pressure, o

so

p

pp is termed relative

lowering of vapour pressure. According to Raoult's law, the relative

lowering of vapour pressure of a dilute solution is equal to the mole fraction

of the solute present in the dilute solution.

Now, if 'n' moles of solute be dissolved in N moles of the solvent, the

mole fraction of the solute will be = Nn

n

According to Raoult's Law, o

so

p

pp =

Nn

n

(i)

If a solution is made by dissolving w.g. of the solute (molecular

weight m) in Wg of the solvent (molecular weight M) the mole fraction of

the solute will b,

303

M

W

m

w

m/w

As in dilute solution the amount of solute is very small, m

w can be

neglected in the denominator as compared to W/M, the equation (i)

becomes.

o

so

p

pp =

m

w.W

M

So, measuring relative lowering of vapour pressure, the molecular-

weight of the solute can be determined.

Relative lowering of vapour pressure is determined experimentally by

Ostwald and Walker method.

Elevation in Boiling Point (Ebullioscopic method) and Depression of

Freezing Point (Cryoscopic method)

We know, the boiling point of a liquid is the temperature at which its

vapour pressure is equal to the atmospheric pressure.

Similarly, the freezing point is defined as the temperature at which the

vapour pressure of its liquid is equal to the vapour pressure of the

corresponding solid.

As the vapour pressure of a pure solvent is higher than that of the

solution hence the elevation in boiling point and depression in freezing point

will be proportional to the mole fraction of the solute.

If b

T = Elevation in B.P. and

304

f

T = Depression in F.P

Then,

oo

so

bp

P

p

ppT

(where p =- po – ps)

and o

p

PT

f

Now, according to Rooult's Law

W

M

m

w

p

P

o

so,

W

M

m

wPT

oband

W

M

m

wPT

of

As for the pure solvent po and M are constant, therefore

w.m

w

w

i.

m

wT

b or

W.m

wkT

bb ………………(ii)

W.m

wT

f or

W.m

wkT

ff ………………(iii)

where, kb is pulsation constant, and

305

kf is depression constant

When, 1m

w (i.e. one mole of nonvolatile solute is dissolved in 1 g of

the solvent)

bb

kT (i.e. kb is equal to the elevation in B.P. when 1 mole of

solute is dissolved in 1 g of the solvent)

and, ff

kT (i.e. kf is equal to the depression in F.P. when 1 mole of solute

is dissolved in 1 g of the solvent)

For practical purpose, in place of kb or kf, kb and kf are used, where.

kb = Modal elevation constant = The elevation in B.P. when 1 mole of solute

is dissolves in 1000 g of the solvent.

Thus, 1000 kb = kb

Similarly.

kf = Model depression constant = The depression in F.P. when 1 mole

of solute is dissolved = 100g of the solvent.

Thus, 100 kf = kf

So (ii) and (iii) relation will be-

bT = Molality x kb or = Wxm

wKb.1000

fT = Molality x kf or = Wxm

wK f .1000

306

Elevation in B.P. is experimentally determined by Landsberger

method and depression in F.P. is experimentally determined by Beekmann's

method. In both of these methods Beckmann thermometer is used to record

small change in temperature. Knowing the value of fb TorT and the

strength of the solution molecular weight of the solute (m) can be

determined.

Osmotic Pressure

For dilute solutions, according to Van't Hoff theory, the equation PV

= nST holds good.

If wg of solute (molecular weight m) be present in V'liters of solution,

then

m

wn and V = V

1

Thus, the equation PV = nST becomes,

STm

wPV .1

or 1PV

TSwm

where, P = Osmotic pressure, T = absolute temperature

S = Molar Solution Constant = 0.082 lit atm. K-1

mol-1

Knowing the value of P experimentally, the value of m (molecular

weight of the solute) can be determined.

Osmotic pressure is determined using Berkeley and Hartley method.

307

6.4.2 Extent of Hydration: Solubility

As has been pointed out earlier the solubility of a polymer is a

function of molecular structure, composition, and molecular weight.

Polar polymers are usually more soluble in polar solvents, e.g. poly

vinyl alcohol, water, whereas non-polar polymers are more soluble in non-

polar solvents, e.g. polystyrene in toluene.

In crystalline polymers the intermolecular crystalline forces must be

overcome by the solvent. Cross-linked polymers swell in a compatible

solvent rather than dissolve. The rate of solution decreases with increasing

molecular weight and increasing length of side chain launching.

As a matter of fact polymer solutions exhibit large deviations from

their limiting infinite dilution behavior. Hence, not only, all the experiments

are carried out at low concentrations but they are invariable extrapolated to

infinite dilutions. In the case of chain molecules, a polymer molecule is

assumed to have a symmetrical statistical distribution of chain elements

about a centre of gravity and the volume occupied by this distribution is

many times the actual molecular volume. Thus the volume over which an

individual polymer molecular exerts its influence depends of the chain

length and on the interaction between the polymer and the solvent. Hence,

the solution taken for physical measurements should be so dilute that each of

the molecule occupies separate portion on the volume.

Sedimentation Equilibria

On ultracentrifuging the polymer solution, several boundaries are

observed revealing the presence of different components in the polymer.

308

This method is useful only where there is sufficient difference in the

molecular weights. This fact is used in Sedimehtaism equilibrium method

for molecular weight determination.

In this method at the equilibrium stage rate at which the solute is

driven outwards by the centrifugal force is equal to the rate at which it

diffuses inwards due to concentration gradient.

The sedimentation rate = Cw2 x M (1-vp) (I/f) .................(1)

The diffusion rate = dx

dc

f

KRT .................(2)

RT

dxw)Vp1(M

C

dc 2 .................(3)

Integrating (2)

)xx(w)Vp1(

)lcc(inCRTM

2

1

2

2

2

12w

.................(4)

This method requires the time for equilibrium which was found to be

too long with substances with molecular weight greater than 500. Shortly

after the centrifuge is brought to speed a determination of concentrations at

the top meniscus and bottom of the cell, gives the equilibrium values.

6.5 HYDRODYNAMIC METHODS

6.5.1 Diffusion

The transport of molecule in the absence of bulk flow is called

diffusion. It is the directed thermal motion of molecules or fine particles

from places of high concentrations. This random movement was observed by

Brown and is known as Brownian movement. This is brought about from the

309

bombardment of the dispersed particles by the molecules of dispersion

medium.

Ficks has shown that if an amount dw i.e., the number of grams of

macromolecules is transferred across the boundary of area A in the direction

X in time dt, it is proportional to the concentration gradient dx

dc.

dx

dcDA

dt

dw

where D is known as diffusion coefficient.

The force that derives the molecules to more dilute region is given by

dx

dc

c

1

N

RTf

This is balanced by the frictional force exerted by the viscous

solution. Stokes found that for a spherical molecule of radius r the force for a

viscous flurid of viscosity is given by

dt

dxr6f

dx

dC

C

1

N

RT

dt

dxr6f

or dx

dC

r6

1

N

RT

dt

dxC

dx

dC

r6

1

N

RT

dt

dw

310

Nr6

RTD

The volume of the spherical molecules is Ndr 3

3

4 . Therefore

the molecular weight is given by Ndr 3

3

4 where d is the density,

NdrM 3

3

4

If the is molecule is non-spherical, then

factorasymmetricf

f

D

D

pherical

phericalnon

sphericalnon

spherical

Fick's second law state that

2

2

dx

cdD

dx

dc .................(5)

The rate at which the boundary between the solution of the polymer

and the solvent get blurred is measured and then D can be calculated.

Integrating Eq. 5 we obtain Wiener's equation

Dt4

xexp

DT4

C

dx

dc 2

o

(9.136)

where Co is the concentration of the solution in glcm3, t is the time for

the beginning of diffusion and x is the distance of the gradient from the

boundary (Fig. 6.8)

There are two methods of determining D, namely

311

(1) Free boundary spreading, and (2) Diffusion through porous plate.

Rectangle cells (Fig. 6.7) are used to study free boundary diffusion. A

sharp boundary can be easily formed by using a sliding joint to superimpose

the solvent on the solution. The boundary spreading is observed by

refraction changes in a polarization diffusion meter at certain levels x at

infinite time interval (Fig. 6.8).

Dt

A

dx

d

dx

dnn

4maxmax

where n is the refractive index and A is the area under curve.

Fig. 6.7 (a)

From equation 6 can be seen that D depends on concentration,

Extrapolating to zero concentration, Do can be obtained.

For a wide range of polymers

b

oMKD

312

where K is a constant for the given polymer solvent system and b is a

parameter.

Lamm has designed micro apparatus to measure D which requires

only I cc of solution.

6.5.2 Sedimentation Velocity Method

The macromolecules having a large size and heavy mass, settle out

of dispersion under the gravitational force. The force F causing

sedimentation of spherical particles is given by

dt

dxr6g)pp(r

3

4F 3

where r is the radius of the particle, p & po are the densities of the

particle and suspension medium respectively, is the viscosity of the

solution and dt

dxis the velocity of sedimentation

9

)(2 3 gpp

rdt

dxo

Since the retarding force is equal to the sedimentation velocity

gpp

dt

dx

ro)(2

9

The radius of the particle can be determined by the path it traverses in

a definite time. It was observed that a particle of radius 10-7

mm with a

density of 2.5 g per cm3 will take about 100 years to settle down. Wiegner,

Kelly and Stamm have designed equipment to measure the sedimentation

velocity of colloidal particles.

313

Svedberg and others

developed analytical ultracentrifuges to

determine the velocity of sedimentation. A particle of mass m at a distance x

from the centre of rotation will experience a centrifugal at force, fc. given by

-

fc = m x w2

Fig. 6.7 (b) Fig. 6.8:

where w is the angular velocity in radian per second, i.e. 2 times the

number of revolutions per second

xwppvdt

dxf

o

2)(

according to Stokes law -

dt

dxrxwppv

o6)( 2

where r is the radius of a given macromolecule in a given solution is

its sedimentation coefficients

r

gvpm

xw

dxldts

6

)1(2

on integration -

314

)(

12

2

12

ttw

xlxInS

The value of w2x comes out to be 2.36x10

8 cm sec

-2 in a centrifuge

with 60,000 rpm and at a distance of 6 cm which is 240,000 times greater

than the acceleration in the earth's field.

If one mole of the substance is sedimenting, then

M = vpN

or dt

dxrxw

p

pM

o

61 2

p

Vp

xw

dtldxM o

o

1

12

where V is the partial specific volume of the dispersed phase

pand is the viscosity and density of the polymer solution and oo

p, is the

density of the medium.

)1( VpD

RTSM

where D is the diffusion coefficient. For precise measurements S, D

and V are extrapolated to infinite dilution. This method takes very long time.

The solution to be studied is placed in a cell with thick quartz windows. A

beam of light is passed through the solution placed in the cell of

ultracentrifuge.This beam of light falls on a photographic plate placed

beyond the cell when the cell is rotated at velocity of 50,000 rpm, the

interface between the solution and the solvent gradually shifts with the

315

sedimentation of the particles and the light is absorbed to different extent at

different heights of the cell. By measuring the optical densities at different

time intervals, the sedimentation velocity can be measured. The curve of

distribution of concentration gradient along the height of the cell at different

time integrals is plotted. Then the curve between lnx and t is plotted which

comes out to be a straight.

The slope of the curve gives the sedimentation constant 4.5 extra

polating lasting it to infinite dilution So is obtained

So = K3 M(1-b)

where Ks is a constant for a given polymer solvent and b has the same

interpretation as in diffusion. The values of b+K3 are given in the literature.

Thus M can be calculated.

6.5.3 Viscosity

A shear stress when applied to a body displaces a plane in the body

parallel to itself relative to other parallel planes in the body. The fluids begin

to flow as soon as the stress is applied. Even solids somewhat flow when the

stress is maintained for a long time. This slow flow of solids is called creep.

Under high stress creep passes over into plastic deformation of solids.

Silicone polymer gives bouncing putty which is a hybrid of solid and liquid

in regard to its flow properties. Viscosity is a measure of resistance that a

body offers to flow. Maxwell showed that the relaxation time t is given by

316

K

t

where is the shear viscosity and K is shear elasticity, liquid of

complex structures display considerable elasticity due to coiling and

uncoiling of molecular chains.

Streamline Flow: If the speed of the flow is not very fast, and the liquid

moves under a pure shearing motion it is called the laminar or streamline

flow. When the liquid moves with high velocities, the flow becomes

turbulent. The Reynolds number RN attains a value 103 to 10

4

PvaR

N where a gives the dimensions of the flowing object.

avrR

N where r is tube radius and v the average velocity of the

fluid.

Restricting to isotropic bodies, let us consider the plane X having

planes X1 & X

11 above and below at a distance l, known as mean free path.

Let n be the number of molecules of mass m per unit volume. Let dv be the

difference in velocities of two layers as a distance dx. Therefore the net rate

of upward flow of momentum through any given plane is given by

dx

dv

dt

dv1vmn

3

1

where v is the root men square velocity, The rate of down flow and

transfer of momentum per unit area of the plane x from above is

317

dx

dylvmn

6

1 . This change of momentum is balanced by a force f acting on

area A of the moving plane.

dx

dylvmn

A

f

3

1

where is called the coefficient of viscosity

lvmn3

1

In the case of gases

2

1

3

M

RTv

and

RT

pMmn

11sec3

3

1

cmgl

M

RT

RT

pM

lRT

Mp

3

1

2

251072.33

1

pd

RT

RT

Mp

2251096.2 dlMRT

318

where d is the molecular diameter and p is the pressure and N is

Avogadro's number. Poise is the unit of viscosity. If a force of 1 dyne cm-2

causes a plane to slide past a parallel surface 1 cm-2

apart with a velocity of 1

cm sec-1

, then the viscosity will be 1 poise.

6.5.4 Electrophoresis and Rotational Motions

Each protein has a characteristic isoelecric point, and this property can

be used in the separation of proteins. In the technique called isoelectric

focusing, proteins are subjected to electrophoresis on a pH gradient. Each

protein moves until it reaches a pH equal to its individual isoelectric point.

At that moment, migration in the electrical field stops because the net charge

of the protein is zero.

The techniques of isoelectric focusing and SDS polyacrylamide gel

electrophoresis have been combined to produce two-dimensional separation

of proteins. Several hundred cellular proteins can be resolved from one

another. This technique is increasingly used in cell biology, and its great

resolving power is due to the use of two independent properties of proteins.

The proteins are first separated by isoelectric focussing (This is the

dimension) which separates proteins according to their charge (isoelectric

point). The proteins are subsequently separated by electrophoresis (this is the

second dimension) in polyacrylamide gels containing SDS, which separates

proteins according to their size (molecular weight). This technique results in

a series of spots distributed throughout the polyacrylamide gel (if the same

property of proteins had been used in both dimensions, the spots would be

distributed along a diagonal).

319

When the detergent sodium dodecyl sulfate (SDS) is used in

electrophoresis, the proteins are separated mainly according to their

molecular weight. This is because SDS binds to the proteins, giving them

large numbers of negative charges due to the sulfate. Thus, most of the

proteins charges will come from the SDS, minimizing the role of charge

differences between individual proteins (differences which would otherwise

affect electrophoretic mobility), and all the proteins migrate according to

their size. The larger proteins move more slowly than the smaller ones

because they encounter more resistance when traversing the molecular

proess within the polyacrylamide gel used for electrophoresis. SDS

electrophoresis is widely used as a method for determining molecular

weights of proteins.

In summary, the vapous pressure methods are applicable to vapours

that follow perfect gas equation. In this case also Victor Meyer's method is

of higher precision. These can be applied to oligomers only whose critical

temperature and critical pressure are known.

Among the ebulliometric and cryoscopic methods, the cryoscopic

methods are more precise. Let us suppose a solution of concentration o 1 g /

100 ml. of solvent which at M = 100 correspond to a number of moles n =

0.01. If cryoscopic constant is 5, then the depression in freezing point will be

0.05oC. If molar mass is M = 10

6, the value of n=10

-6. Hence Tf = 5 x 10

-6.

No existing method can measure such insignificant changes in temperature.

The conventional cryoscopic method can determine the molecular mass from

15,000 to 30,000. The osmometry can be used for determining molecular

masses from 104 to 10

6.

320

Viscometric methods are not recommended for determining absolute

molecular masses but only changes in molecular masses during various

processes (polymerization, degradation, etc.)

The method of sedimentation in the ultra-centrifuge is an absolute

method for measuring the molecular mass of a polymer.

The light scattering method is more precise and makes possible to

calculate the value of the mw molecular mass of polymers without

assumption of the shape of macromolecules in a solution.

CHECK YOUR PROGRESS-2

Notes: (1) Write your answers in the space given below.

(2) Compare you answers with those given at the end of the unit.

(a) (i) Depending on the method of determination, different average

molecular weights are obtained. These are

(a) .........................................

(b) .........................................

(c) .........................................

(d) .........................................

(ii) In the case of monodispersed systems different average

molecular weights are related as : ......................................

(iii) The molecular size can be determined by ........................,

..................................., and ............................ .

(b) (i) The molecular weights, may be determined, using colligative

properties such as -

(a) .........................................

(b) .........................................

321

(c) .........................................

(d) .........................................

(ii) Hydordynamic methods include –

(a) Diffusion (b) Sedimentation (c) Ve

(iii) .................... electrophoresis is widely used as ................... for

determining molecular weights of ...............

6.6 LET UP SUM UP

By going through this unit you must have achieved the objectives laid

down at the start of the unit.Let us sum up what we have discussed so far:

* Biopolymer characterization is a must for any worker dealing with

these. The information is obtained for the selection of polymers for

specific properties. Special techniques have to be employed for

composites and polymer blends. NMR, IR, gas chromatography,

mass spectroscopy, UV, luminescence and electron spectroscopy are

important.

* Polymer chain interaction may result in an ordering of chains in

crystalline or amorphous phases. The small changes in size and

distribution of these ordered regions may bring about differences in

physical and other properties in different samples.

* It is possible for the polymers to have the average molecular weight

but different molecular weight distribution (MWD).

322

* Statistically, we can express the average in terms of number or

weight. Consequently, the molecular mass of a polymer is expressed

as number average mass ( nM ) or weight average molecular mass

( wM ).

The ratio of the weight and number average molecular masses

( wM \ nM ) is called 'Poly-dispersity index' (PDI). In natural

polymers which are generally mono dispersed, the PDI is unity i.e.

wM = nM .

* Mn is determined by employing methods which depend upon the

number of molecules present in the polymer sample, viz colligative

properties like osmotic pressure, depression in freezing point and

elevation in boiling points. On the other hand methods such as light

scattering and ultra-centrifuging depend on the mass of the

individual molecules and yield weight average molecular mass.

* The solubility of a polymer is a function of molecular structrue,

composition and molecular weight.

In crystalline polymers the inter molecular crystalline forces

must be overcome by the solvent. The rate of solution decreases

with increasing molecular weight and increasing length of side chain

launching.

.)lnso(.)lnso(.)lnso(

STHG

The dissolution of a polymer as favored if H < TDS.

323

* Osmosis is defined as the forces of net diffusion of water molecule

from a dilute solution or pure water (solvent) itself to a more

concentrated solution, when both are separated by a semi permeable

membrane.

The membrane allows the water to diffuse but not the solute.

* The hydrostatic pressure which exactly balances the osmotic influx

of water from pure water to concentrated solution is called the

osmotic pressure of that solution.

* Calcium helps in the release of acetylcoline from the storing vesicles

present in the nerue ending. Calcium ions neutralize the negative

charge of myosin which then combines with negative charged actin

and thus the concentration takes place. Calcium ions (Ca++

) also

activate the myosin AT Pase which in turn breaks down the ATP to

supply energy required for muscular contraction.

* The theories of muscle contrition are (a) Ratchet theory and (b)

Electrostatic solenoid theory.

Immediate source of the energy required for muscular

contraction is ATP provided by oxidative phosphorylation but not at

a sufficient rate to sustain muscle during bursts of intense activity.

The presence of an enzyme myokinase which catalyses the transfer

of a high energy phosphate from one molecule of ADP to another to

from ATP and AMP.

AMPATPADP2Myokinase

* Molecular weights are determined either directly or in solution.

324

The method for direct determination of molecular weights can only

be applied to gases or volatile liquids and solids.

However, molecular weights of biopolymers are generally

determined by solution methods. The important methods involve use

of colligative properties viz (i) lowering in the vapour pressure (ii)

elevation in the boiling point (iii) depression in the freezing point

and (iv) osmotic pressure for the determination of molecular weight.

* The hydrodynamic methods for determination of biopolymer

molecular weight, include (i) diffusion method,(ii) sedimentation

method and (iii) viscosity method.

* However SDS electrophoresis is widely used as a method for

determination of molecular weights of proteins.

6.7 CHECK YOUR PROGRESS : THE KEY

(a) (i) average molecular weight

molecular weight distribution

(ii) decreases

molecular weight

increasing side chain

(iii) membrane

water

diffuse

solute

(b) (i) the negative charge

myosin

actin

325

constraction of muscles

(ii) (a) Ratched theroy

(b) Electrostatic solenoid theory

(iii) myokinase

high energy phosphate

ATP

AMP

2(a)(i) (a) Number average molecular weight n

M

(b) weight average molecular weight w

M

(c) Velocity average molecular weight V

M

(d) Z-average molecular weight z

M

(ii) n

M = w

M = V

M = z

M

(iii) degree of polymerisation

radius of gyration

hydrodyanamic volume

b(i) (a) Lowering in the Vapour pressure

(b) Elevation in boiling point

(c) Depression in freezing point

(d) Osmotic pressure

(ii) (a) Diffusion

(b) Sedimentation

(c) Viscolity

(iii) SDS

method

326

proteins

327

M.Sc. (Final) Chemistry

PAPER –III : BIOINORGANIC, BIO ORGANIC & BIOPHYSICAL

CHEMISTRY

BLOCK – III

Unit-7 : Enzymes

Unit-8 : Enzyme Action

Unit-9 : Co-Enzymes

Unit-10 : Biotechnological Application of Enzymes

Author – Dr. Purushottam B. Chakrawarti

Dr. Aruna Chakrawarti

Editor - Dr. Anuradha Mishra

328

SUMMARY

Enzymes are the chemical catalysts that control all biochemical

reactions. These are complex protein molecules present in living cells.

Enzymes are classified into six main classes. They are named by adding

suffix 'ase' to the name of the substrate. Enzymes are isolated from a cell and

purified using various methods. According to most accept acceptable

hypothesis the enzyme combines with substrate to form intermediate enzyme

substrate complex, which then breaks down into product and enzyme.

Mechanism of catalytic activity of an enzyme is studied using kinetic

procedures, analysis of active centres of enzyme and comparative x-ray

studies of complexes. The enzymes whose mechanism of action is

extensively studied include chymotrypsin, ribonuclease, lysozyme and

carboxypeptidase.

Some enzymes are simple proteins while some are conjugated

proteins. In such enzymes the non-protein part is called prosthetic group or

enzyme and the protein part is called apoenzyme. Most of the coenzymes are

nuclestides and are composed of vitamins. Same important coenzymes are

A-pantothenic acid, B1–thiamine pyrophosphate, B6–pyridoxal phosphate,

Niacin, riboflavin, Lipoic acid and B12. Enzyme models are synthetic models

which have two one or more properties of enzyme systems. Biomemetic

chemistry is used to show chemical processes taking place during a

biochemical reaction, using synthetic models.

The synthetic molecules which have catalystic activity similar to a

natural enzyme are known a 'Synzymes' or syntehtic enzymes. Several

industrial processes such as food, diary. sugar, soft drink, brewery, textile,

leather and pharmaceuticals use immobilised enzymes. The use of enzymes

in drug design and in immobilised enzymes. The use of enzymes in drug

design and in enzyme therapy has opened new doors for making human free

from several diseases.

329

7. ENZYMES

Structure

7.1 Introduction

7.2 Objectives

7.3 Nomenclature and Classification

7.4 Chemical and Biological Catalysis

7.5 Extraction and Purification

7.6 Properties of Enzymes

7.6.1 Catalytic Power

7.6.2 Specificity and Regulation

7.7 Mechanism of Enzyme Activity

7.7.1 Fischer's Lock and Key Hypothesis

7.7.2 Koshlands Induced Fit Hypothesis

7.7.3 Identification of Active Site :

Use of Inhibitors

Affinity Lebeling

Enzyme Modification : Site directed mutagenesis

7.8 Enzyme Kinetics

7.8.1 Michaelis – Menten Plots

7.8.2 Lineweaver Plots

7.83 Reversible and Irreversible Inhibition

7.9 Let Us Sum Up

330

7.10 Check Your Progress : The Key

331

7.1 INTRODUCTION :

Enzymes are the chemical catalysts that control all biochemical

reactions in every living thing, from viruses to man. Breathing, digestion,

heart action, formation of body tissues, movement of muscles all these and

many more depend on enzymes. In short we can say that without enzymes

there is no life.

Every cell synthesise its own enzymes and a single body cell contains

as many as 1,00,000 enzymes, each directing a specific reaction, each

coming into play at the right moment and place. More than 1,00,000

different types of enzymes have been identified most of which are colourless

solids, soluble in water or dilute solution, but some are blue, green or

greenish brown.

Most of the enzymes, produced by a cell, function within that cell and

hence are called endoenzymes (intracellular) but some enzymes are liberated

by living cells and catalyze reactions in the cell's environment, such

enzymes are known as exoenzymes (extracellular). If an enzyme is secreted

in a form which act upon the substrate as such i.e. without undergoing prior

modification in structure, it is known as the zymase or active form of the

enzyme. Intracellular enzymes belong to this class. On the other hand, in

some cases the enzymes exist as inactive precursors called zymogens which

are activated on coming into contact with an activating agent. For example,

pancreas and pancreatic juice contain the zymogen trypsinogen which is

itself inactive but is converted into active trypsin on coming into contact

with previously formed trypsin or with the enteropeptidase, enterokinase of

duodenal mucosa. By producing zymogens the organism is protected from

332

the action of active enzymes in situations where their activity might prove

injurious. Most cases of pre-enzyme activation involve the removal of an

inhibitory or blocking peptide moiety from the pre-enzyme molecule.

Enzymes are complex protein molecules present in living cells, where

they act as biological catalysts and bring about chemical changes in

substances. Virtually all the chemical reactions occurring in

microorganisms, plants and animals proceed at a measurable rate as a direct

consequence of enzymic catalysis. Without enzymes there can be no life.

Enzymes can change plants and animals in a precise and often remarkable

dramatic fashion. In the hands of expert biotechnologists enzymes become

the tailor's scissors and the surgeon's scalpel. Although enzymes are formed

only in living cells, they can be separated from the cells and can continue to

function in vitro. This unique ability of the enzymes of perform their specific

role in an isolated system formed the basis of an ever-incrasing use of

enzymes in industrial processes (medicine, research and rDNA technology)

that are collectively known as enzyme technology.

Enzyme technology involves the production, isolation, purification,

use in soluble form and finally immobilisation of enzymes and also their role

in various industries including dairy, baking, brewing, textile, food

production, leather, pharmaceutical and medicine. In addition, enzyme

technology has immense importance in development of biosensors that have

tremendous role to play in world economy.

HISTORICAL BACKGROUND

W. Kuhne coined the word ‘enzyme’ in 1878 from the Greek term

meaning yeast. Earlier enzymes were referred to as ferments because their

333

action was similar to yeast fermentation. Some of the chronological events

in the field of enzyme study are listed in table 6.1, indicating that during the

succeeding years enzymology has developed apace.

Table 7.1 : Chronology of enzyme study

1833 Payen and Persoz achieved alcohol precipitation of thermolabile

'diastase' from malt.

1835 Berzelium put forth the concept of catalysis.

1850 Wilhelmy studied quantitative evaluation of rates of sucrose

inversion.

1878 Kuhne investigated trypsin-catalysed reaction, and introduction

of the word 'Enzyme'

1890s Fisher suggested the 'Lock and Key' model for enzyme action.

1898 Duclaux suggested nomenclature for the enzymes with substrate

plus suffix 'ase'

1906 Harden and Young studied coenzymes (NAD)

1913 Michaelis and Menton proposed mathematical modelfor kinetic

theory of enzyme action.

1926 Summer isolated enzyme urease in crystalline form.

1937-39 Cori and Cori situation muscle phosphorylase.

1940 Beadle and Tatum put forth 'one gene – one enzyme' hypothesis.

334

1948 Pauling. Transition state theory of enzyme action.

1951 Pauling and Corey. Secondary structure of enzyme.

1953 Koshland. Induced fit hypothesis

1952 Sanger, Amino acid sequence of protein hormone, insulin.

1956 Sutherland. Cyclic AMP – Second messenger.

1961 Jacob, Monod and Changeux. Allosterism.

1986 Cech. Ribozyme-RNA with catalytic activity.

1994 Cech and Uhelnbeck, Hammer headed ribozyme to fight viral

diseases.

1997 Tang and Breaker. Rational design of allosteric ribozymes.

1999 Robertson and Ellington. In vitro selection of an allosteric

ribozyme.

2000 Bergman et al. Kinetic framework for ligation by an efficient

RNA ligase ribozyme.

2001 Seetharaman et al. Ribozyme in ELISA – like assay.

2002 Iyo et al. Allosterically controllable maxizymes for molecular

gene therapy.

2003 Vaish et al. Half-ribozyme activated by hepatitis C virus (HCV)

sequences.

335

7.2 OBJECTIVES

The main objective of the unit is to discuss nature, type and activity of

enzymes. After going through this unit you would be able to :

describe what are enzymes and how they are named and classified,

explain extraction and purification of enzymes,

discuss properties of enzyme,

explain mechanism of enzyme activity and discuss enzyme – kinetics.

7.3 NOMENCLATURE AND CLASSIFICATION

Enzymes are the largest and most specialised class of protein

molecules that catalyse a reaction in which a substrate (S) is converted to

product (P) through the formation of an intermediate enzyme-substrate (ES)

complex. On the basis of the type of reactions that they catalyse, enzymes

are classified into six main classes.

When enzymes were first discovered they were given various

unsystematic names by their discovers, such as pepsin, trypsin, ptyalin, and

zymase. Later on enzymes were named by additing suffix 'ase' to the name

of the substrate (the substrance on which enzyme acts), e.g., esterase which

acts on (hydrolyses) ester; amylase which acts on starch (amylum), urease

also denotes urea, etc. Furthermore, the name of some enzymes also

denotes the type of reaction along with the substrate, e.g., lactic acid

dehydrogenase indicates that the enzyme catalyses the dehydrogenation

(removal of hydrogen) of lactic acid. However, some enzymes were named

before these general rules were formulated and such enzymes have retained

their trivial names, e.g., emulsin, pepsin, trypsin, etc.

336

There is no perfect method for the classification of enzymes.

However, the most comprehensive system for the classification of enzymes

was devised in 1961 by the Enzyme Commission of the International Union

of Biochemistry (I.U.B.). The major features of the IUB system for

classification of enzymes are given below.

(i) The enzymes are classified first according to the general type of

chemical reaction they catalyse. There are six important classes.

(ii) Each class is further classified into several sub-classes on the

basis of the type of bond split or formed, chemical group

removed or transferred, or in some cases by a simple sub-

classification of the general reaction type.

(iii) Main classes and subclasses are indicated by index numbers.

(iv) A third figure is sused for more detailed subdivision of the

subclass.

(v) The serial number of the specific enzyme within its own sub-

class is indicated by the fourth figure.

(vi) Thus on the basis of the above points, each enzymes is given a

systematic code number commonly known as enzyme

commission (E. C.) number. For example, 1.1.1.1 is the enzyme

code for alcohol dehydrogenase : the first digit characterizes the

reaction type, the second subclass, the third sub-class and the

fourth digit indicates the particular enzyme named. Thus, E.C.

2.7.1.1 denotes class 2, sub-class 7 of the class 2, sub-sub-class

1 of the subclass 7, and the last digit denotes that the enzyme is

the first in the sub-subclass 1.

337

Although the IUB system is complex, it is precise, descriptive,

and informative. However, since these systematic names are

frequently too long for ordinary use, the trivial names are commonly

used. The trivial names are made up from the name of the substrate,

the type of reaction catalysed and the sufficx 'ase', e.g., alcohol

dehydrogenase whose systematic name is alcohol : NAD

oxidoreductase and enzyme commission (E.C.) number is 1.1.1.1.

The major six classes of enzymes are now discussed in some

detail. The complete classification of enzymes is summarised in table

7.1

1. Oxidoreductases : Oxidoreductases are those enzymes which are

involved in biological oxidations and reductions. This class includes several

subclasses, viz. dehydrogenases, oxidases, oxygenases, hydroxylases,

hydroperoxidases, etc.

AH2 + B A + BH2 (Reduction)

AH2 + O2 A H2O (oxidation)

Dehydrogenases : The dehydrogenases are enzymes that catalyze the

removal of hydrogen from one substrate and pass it on to a second substrate,

i.e. they are not capable of passing the hydrogen directly to oxygen.

Although the various dehydrogenases differ in the structure of the

apoenzyme, they have the same coenzyme viz. nicotinamide adenine

dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate

(NADP+). The reaction of the dehydrogenases may be shown schematically

as below :

Sub AH2 E – N A D+ Sub BH2

338

Sub A E – NADH + H+ SubB

Sub = Substrate, E = Enzyme

The schematic equation shows that the substrate A transfers a

hydrogen atom with its bonding pair of electrons and a proton (H+) to the

enzyme (E) having NAD+ as the coenzyme. The reduced enzyme (E –

NADH + H+) as the coenzyme. The reduced enzyme (E-NADH+H

+) in turn

transfers its hydrogen to another substrate B.

In NAD+ or NADP

+, the nicotinamide moiety serves as the oxidizing

agent or hydrogen carrier.

where R=Rest part of the NAD+ or NADP

+ molecule.

Oxidases: Oxidases are the enzymes which catalyze the removal of

hydrogen from a substrate and pass it directly to oxygen. Again like

dehydrogenases, there are further several classes of oxidases differing from

each other in the structure of the apoenzyme but usually have the same

coenzyme, which, in this case, is flavin adenine dinucleotide (FAD). The

oxidation of a substrate involving an oxidase may schematically be shown as

below.

339

In oxidases, the flavin moiety of the coenzyme FAD serves as a

hydrogen carrier in the following way.

Oxygenases: This subclass of enzymes catalyze the incorporation of

oxygen directly into the substrate. These enzymes require the presence of

metal ions, such as iron or copper.

2. Transferases : These enzymes catalyse the transfer of a group (X)

from the substrate to another, i.e.,

AX + B A + BX

These enzymes are further classified into various sub-classes on the basis of

the nature of the transferring group(X), viz.,—NH2 group (transamination),

phosphate group (transphosphorylation), etc.

3. Hydrolases : The hydrolases are those enzymes which cataly.e

hydrolysis, that is, the direct addition of water molecule (s) takes place

across the bond, which is cleaved. The hydrolases are divided into several

sub-classes on the nature of the group or bond being hydrolysed, viz., ester,

ether, peptide, acid anhydride, glycosyl, C—C, or C—halide bonds. Thus

the various sub-subclasses of this class are esterases, etherases, peptidases,

glycosidases, esterases, phosphatases, thiolesterases, etc.

A – B + H O H A H + B O H

340

The names of these enzymes are usually the name of the substrate

followed by—ase, e.g., penicillinase, urease, dipeptidase, etc. Some

hydrolases have names which do not indicate the type of reaction catalysed

and have unusual endings, e.g., lysozyme and the many peptidases which

end with-in, e.g., chymotrypsin, rennin and papain.

4. Lyases : The lyases are a smaller class of enzymes that catalyze the

removal of a small molecule from a larger substrate molecule. Further since

the ractions are reversible, lyases may also be considered to catalyze the

addition of small molecules to the substrate molecule.

X Y

| |

C C - C=C + X – Y or A = B + x – y AX – BY

These are further classifed on the basis of the linkage being attacked,

viz., C – C, C – O, C – N, C – S, and C – halide bonds. Examples of this

group include aldolase, fumarate hydratase, pyruvate decarboxylase and

enolase.

5. Isomerases : This class includes all enzymes which catalyse

isomerization, i.e., interconversion of optical, geometrical, or positional

isomers. The great variety of enzymes in this group necessiates a sub-

classification based largely on the types of reactions involved, viz.,

racemases, intramolecular transferases, etc.

A A'

6. Ligases (or Synthetases) : These enzymes catalyse synthesis

reactiosn by joining two molecules coupled with the breakdown of a

pyrophosphate bond of adenosine triphosphate, ATP to adenosine

341

diphosphate, ADP. Formation of malonyl – CoA from acetyl – CoA in the

presence of acetyle – CoA carboxylase as catalyst is an important example.

The enzyme requires biotin as a coenzyme.

A + B + ATP + H2O A – B + AD P + Pi

Table-7.1 : Classification of Enzymes

Main Class and Sub-class Example

1. Oxidoreductases

1.1 Acting on the CH – OH group of

donors

1.1.2 With NAD or NADP as acceptor Alcohol dehydrogenase, lactate

dehydrogenase

1.1.3 With O2 as acceptor Glucose oxidase

1.2 Acting on the CHO or CO group

of donors

1.2.1 With NAD or NADP as acceptor Glyceraldehyde-3-P

dehydrogenase

1.2.3 With O2 as acceptor Xanthine oxidase

1.3 Acting on the CH-CH Group of

donors

1.3.1 With NAD or NADP as a

acceptor

Dihydrouracil dehydrogenase

1.3.2 With a cytochrome Acyl-CoA dehydrogenase

1.4 Acting on the CH-NH2 group of

donors

1.4.1 With NAD or NADP as acceptor Glutamate dehydrogenase

1.4.3 With O2 as acceptor Amino acid oxidases

342

Main Class and Sub-class Example

1.9 Acting on the heme groups of

donors

1.9.3 With O2 as acceptor Cytochrome oxidase

1.11 Acting on H2O2 as electron

acceptor

1.11.1 With NAD or NADP Catalase

2. Transferases :

2.1 Transferring C1 – groups

2.1.1 Methyl transferases Guanidoacetate methyl-

transferase

2.1.2 Hydroxylmethyl transferases Serine hydroxymethyl-

transferase

2.3 Acyl transferases Choline acyltransferase

2.4 Glycosyl transferases Phosphorylase

2.6 Transferring amino group (amino

transferases)

Transaminases

2.7 Transfer of P-containing groups Hexokinases

3. Hydrolases

3.1 Acting on esters bonds

3.1.1 Carboxylic ester hydrolases Esterases, Lipases

3.1.3 Phosphoric monoester hydrolases Phosphatases

3.2 Cleaving glycosides

3.2.1 Glycosidases Amylase, -glycosidases

3.2.2 N-Glycosidases Nucleosidases

3.4 Cleaving peptide linkages

343

Main Class and Sub-class Example

3.4.1 -Aminopeptide amino acid

hydrolases

Leucine aminopeptidase

3.4.2 -Carboxypeptide amino acid

hydrolases

Carboxypeptidases

3.4.4 Peptidyl peptide hydrolases

(endopeptidases)

Pepsin, trypsin, chymotrypsin

4. Lyses

4.1 C – C lyases

4.1.1 Carboxyl-lyases Pyruvate decarboxylase

4.1.2 Aldehyde-lyases Aldolases

4.2 C – O lyases

4.2.1 Aldehyde – lyases Fumarase (fumarate hydratase)

4.3 C – N lyases

4.3.1 Ammonia lyases Histidase (Histidine ammonia

lyase)

5. Isomerases

5.1 Racemases and epimerases

5.1.1 Acting on amino acids Alanine racemase

5.1.3 Acting on carbohydrates Ribulose – 5-P epimerase

5.2 Cis-trans isomerases Maleylacetoacetate isomerase

5.3 Intramolecular oxidoreductases

5.3.1 Interconversion of aldoses and

ketoses

Triose phosphate isomerase,

Glucose phosphate isomerase.

6. Ligases

6.1 Forming C – O bonds

344

Main Class and Sub-class Example

6.1.1 Amino acid – RNA ligases Amino acid – activating

enzyme

6.2 Forming C – S bonds

6.2.1 CoA Ligase Amino acid activating enzyme

6.1 Forming C - S bonds

6.3.1 Acid – ammonia ligases Glutamine synthetase

6.3.2 Acid-amono acid ligases Peptide synthetase, glutathione

synthetase

6.4 Forming C – C bonds

6.4.1 Carboxylases Acetyl – CoA carboxylase

7.4 CHEMICAL AND BIOLOGICAL CATALYSIS :

Enzymes are biocatalyst, they catalyse biochemical reactions. Most of

them are produced by a cell; while chemical catalysts are the substances

added into the reaction mixture in small quantities generally from outside.

Without enzymes there can be no life, however in a chemical reaction

absences of a catalyst generally the reaction takes place at a slow rate.

Unlike the catalysts employed in the chemical laboratory which can

withstand quite severe conditions, the activity of enzymes is markedly

affected by several factors, viz. temperature, pH (hydrogen ion

concentration) and concentration of other ions. They exhibit highest activity

under optimal conditions. Each of these effects is discussed in a some

details.

The effect of temperature : With certain exceptions, the rates of

chemical reactions are increased as the temperature is raised.

345

As we know, the increase of reaction temperature actually increases

the total energy of the chemical system with the result its activation energy

is decreased which in turn increases reaction velocity.

Like most chemical processes, rise in temperature causes increase in

reaction rate in enzyme-catalysed reactions. However, this happens so only

upto a certain temperature commonly known as optimum temperature

(usually in the range of 40oC – 60

oC) above which enzymes, being

proteinous in nature, undergo denaturation with loss of activity which in turn

is due to loss of secondary and tertiary structure of the protein moiety. As

the enzyme is inactivated the reaction which it catalyses slows down and

ultimately stops.

Thus optimum temperature of an enzyme may be defined as the

temperature at which its (enzyme) activity is maximum. The exact value of

the optimum temperature depends upon the nature of the individual enzyme.

The effect of pH : The reaction velocity of an enzymatic reaction

very much depend upon the pH of the medium. For most of the enzymes,

there is a range of pH (normally 4-9) values outside which the enzyme will

not function. Within this range of pH there is invariably an optimum pH at

which the enzyme has maximum efficiency in catalysing the reaction.

As mentioned above, the optimum pH generally lies in the range of 4-

9, but occassionally more extreme vlaues are also found.

The explanation of pH effect is based mainly upon two factors.

(a) Ionization of the enzyme, particularly at the active site : Like all

proteins, enzyme molecules possess numerous ionizable groups whose state

346

of ionizability depends on pH. For an enzyme molecule to be active as a

catalyst certain of these groups must be ionized while certain others

unionized. This state of affairs would obviously prevail only within a limited

pH range which would depend on the pKa values of the grops concerned.

(b) Ionization of the substrate, or a co-reagent such as a co-enzyme

: In some cases substrate, like the enzyme is also capable of being ionised.

Thus in such cases also it would be reasonable to assume that only one ionic

form of the substrate molecule might be capable of undergoing the reaction.

This specific ionic substrate would also exist over a limited pH range.

Thus owing to the above two facts, the optimum pH for the reaction

would be the pH at which the ionization states of the substrate and enzyme

were most favourable for the reaction. The influence of pH on enzyme

activity explains why the simple organisms like bacteria flourish only within

a restricted pH range and why complicated mechanisms for maintaining the

required pH of the body fluids operate in the complex organisms like man.

Effect of others ions (Activators) : In addition to H+, which is the

only cation whose concentration is important for all enzymes, certain other

ions (e.g., Mg++

, Ca++

, Mn+-

, Zn++

, Na+ and K

+) are also required for the

activity of many enzymes. Amylases need CI- ions, enzymes turning over

ATP nearly always require Mg++

ions. Several peptidases are activated by

Mn++

, Zn++

, or Co++

. The divalent ions can replace occasionally one another.

The mechanism by which ions exert their influence is known only in a few

cases. Some cations are loosely bound with enzymes, viz. phosphopyruvate

hydratase contains a loosely bound Mg++

removal of which results in

inactivation. In other cases, the ion may combine with the substrate, viz.

347

Mg++

form complexes with nucleoside di-and tri-phosphates in the

enzymatic reactions of the latter compounds.

Effect of substrate concentration : As expected, with a fixed amount

of enzyme, the reaction rate is proportional to the substrate concentration.

But this is true upto a certain point after which the increasing concentration

of substrate does not further increase the velocity of the reaction.

7.5 EXTRACTION AND PURIFICATION :

Various microorganisms like bacteria, fungi and yeast produce

different kinds of enzymes; even the animal tissues and plants are used as

sources of enzymes (table 7.2). Initially plant and animal enzymes were

preferred over microbial enzymes mainly because of safety concern and fear

of contamination by microorganism, toxins etc. But as the demands for

enzymes augmented, the supply of enzymes derived from plant and animal

tissues could not keep the pace. Later on the importance of microbial

enzymes increased because of the advantage of (a) large-scale production by

fermentation, (b) ease in isolation, and (c) involvement of recombinant DNA

technology so that the quantity and quality of enzymes can be modified. For

example, the enzymes rennet (also called chymosin or aspartic proteinase) is

widely used for cheese production; in earlier days, rennet was obtained from

animal tissue like calf stomach. Since it is difficult to maintain a continued

supply of rennet derived from animal tissues, nowadays it is produced on

large scale from the fungal species mucor methei.

Table-7.02 : Biological Sources for Production of Important Enzymes

Source Tissue / Producer

Microorganism

Enzyme Application

Animal Liver Catalase Food

348

Source Tissue / Producer

Microorganism

Enzyme Application

tissue

Pancreas Lipase Food

Pancreas Trypsin Leather

andMedicine

Stomach Rennet Dairy

Plant Malted barley -amylase Food, textile and

brewing

"—" -amylase Brewing

"—" -Glucanase Brewing

Fig Latex Ficin Food

Papaya Latex Papain Meat

Bacteria Baccillus -amylase Starch, textile and

biological

detergent

Baccillus and

streptomyces

Glucose

isomerase

Fructose Syrup

Bacillus Penicillin

amidase

Pharmacy

Bacillus Protease Detergent and

Brewing

Fungi Aspergillus -amylase Baking

Aspergillus and

Rhizopus

glucoamylase Starch

Mucor Meihei Rennet Dairy

Aspergillus Pectinase Fruit Juice

"—" Protease Brewing and

Backing

"—" Cellulase Fruit juices

Rhizopus Lipase Detergents

Aspergillus Lactase Diary

Yeast Sacchromyces Invertase Confectionery

349

Source Tissue / Producer

Microorganism

Enzyme Application

"—" Raffinase Food

For isolation of intracellular enzymes from a cell one of the following

processes may be used :

(i) Crushing the tissue with sand

(ii) Crushing the tissue in a homogeniser

(iii) By crushing the tissue with acctone, lipid of the cell dissolves in

accetone and get separated.

Thus the enzyme is obtained as residue.

The crude extract obtained by above processes is subjected to ,

when small molecules are separated. The nucleic acids are separated by

adsorption on charcoal. Separation of enzymes from the residual extract is

difficult, since it contains many protein molecules, which are more or less

similar in their physical and chemical properties.

For further purification the following methods are used:

(i) Precipitation :

For this generally different concentration of ammonium and

sodium sulfate solution are utilized.

(ii) Solvent Extraction:

For solvent extraction generally acetone or ethanol is used.

(iii) Temperature of pH Denaturation:

This involves heating or acid treatment.

(iv) Differential Centrifugation

350

(v) Electrophoresis

(vi) Selective Adsorption and Elution of Proteins

For this anion exchanger diethyl aminoethyl cellulose or cation

exchanger charboxy methyl cellulose is used. This chromatographic process

separates and purifies enzyme rapidly.

(vii) Use of Sephedex molecular sieve:

Protein molecules of different sizes can be separated easily.

(viii) Affinity Column Chromatography:

Can also purify enzymes.

7.6. PROPERTIES OF ENZYMES

With no exception, enzymes are protein molecules. Some enzymes are

simple proteins, i.e. their molecules consist of only amino acids, where as

others are conjugated proteins. The non-protein part (non-ammo acid of a

conjugated protein or enzyme is known as prosthetic group. The two

fragments (protein + prosthetic group) of some conjugated proteins

(enzymes) can be separated by dialisis and it was seen that dialysis of such

enzyme resulted in the loss of catalytic activity of that enzyme, but the

activity can be regained by mixing the two separated components. This fact

clearly indicates that the dialyzable component is necessary for protein to act

as an enzyme and hence this dializable material (prosthetic group) is termed

as coenzyme, the protein part of a conjugated protein as holoenzyme.

Enzymes are soluble and colloidal in nature and have large macro

molecules. The molecular weight of enzymes varies from 12000 to a lac. It

has been seen generally the molecular weights of most of the enzymes are in

n-times of 17,500 e.g.:

351

Protein Molecular Weight n

Pepsin 35000 2

Catlase 250000 14

Urase 480000 27

Due to large size rate of diffusion of enzyme molecules is generally

very low. The form colloidal sol with water. In addition, there are two

general properties of enzymes, viz. tremendous efficiency and their

remarkable specificity.

(i) Enzyme efficiency: A most important biochemical characteristic of

the living cell is its ability to achieve a large number of rapid chemical

conversions at temperatures usually below 40oC, while the parallel chemical

reactions in laboratory proceed usually at high temperatures.

Chemical

Catalyst

Rapid reaction

Substrate ------------------------- Product

molecule Extremely rapid molecules

reaction

Enzyme

molecules

This characteristic of the living cell is achieved by the catalytic nature of

enzymes. Experiments with enzymes isolated from cells and tissues have

shown that the efficiency of enzymes is greater by as much as own hundred

million (108) times than the chemical catalysts.

The increased velocity of the chemical reaction catalyzed by enzymes

is explained by the fact that the enzymes like other catalysts reduce the

352

energy of activation (Ea) required for that reaction to proceed. In a

Laboratory chemical reaction, the energy of activation is decreased by

increasing the temperature of the reaction. Now since the living organisms

function at temperatures usually below 40oC and owing to the sensitivity of

many cellular materials to high temperatures, the method of heating for

increasing reaction velocity is not appropriate. Thus when energy of

activation of a reaction is decreased, it becomes easier for the reactant

molecules to gain the minimum amount of energy which is required for them

to react, and so the reaction velocity is increased. This is evident from table

7.3 and graph 7.1.

Table : 7.3

Reaction Catalyst Ea in

cal/mole

Relative

reaction velocity

Decomposition of

H2O2

(a) NO

(b) Platinum

(c) Catalase (enzyme)

18,000

12,000

600

1

26,000

>1010

Hydrolysis of

sucrose

(a) Acid

(b) Invertase

26,000

11,000

1

>1011

353

Fig. 7.1. Before a chemical reaction can occur, the reacting molecules must gain a

certain minimum amount of energy – this is the activation energy. The greater the

required activation energy, the slower is the rate of reaction at a given temperature.

(ii) Enzyme specificity: Even more striking than the very high catalytic

activity of enzymes is their specificity. There are three types of enzymatic

specificity, viz. stereochemical specificity, reaction specificity and substrate

specificity. This feature of enzymes can provide an explanation for the

integration and organization of the large number of different chemical

reactions that occur in living cells.

Furthermore, the fact that each enzyme has its own set of specificities

suggests that there must be a larger number of different enzymes. Such in

the case; the number of known enzymes approaches nearly a thousand. In

general, the stereo-and substrate specificities of an enzyme are attributed to

the protein part of the apoenzyme while the reaction specificity is more

attributable to the coenzymes or prosthetic group of the apoenzyme.

354

(a) Stereospecificity: The fact that most of the biological reactions are

extremely specific in the production of stereoisomers will frequently come

across throughout the book. For example, succinic acid is dehydrogenated to

give only fumaric acid and no maleic acid which might also have been

produced during the chemical dehydrogenation. D-Glyceraldehyde-3-

phosphate and dihydroxyacetone phosphate are combined to give D-

fructose-I, 6-diphosphate which is one of the four possible optical isomers

that might have been produced. Enzymes are specific in the oxidation of D-

and L-amino acids. A specific enzyme will oxidise only one of the two

optical isomers. The example that could be cited are almost innumerable.

CH2. COOH HCCOOH

| ||

CH2.COH HOOCCH succinic acid Fumaric acid

CH2OP

|

CO

|

CHO CH2OP HOCH

| | |

HCOH + CO Aldolase HCOH

| | |

CH2OP CH2OH HCOH

|

CH2OP

D-Glyceraldehyde- Dihydroxyacetone Fructore-1, 6-

3-phosphate Phosphate diphosphate

(b) Reaction specificity: Enzymes are specific in the sense that almost

invariably one enzyme catalyes only one of the reaction which is the

substrate can undergo. For examples, oxalocaetic acid (an important

intermediate in metabolism) can undergo several reactions, viz. reduction to

355

give malic acid, decarboxylation to give pyruvic acid, or it can accept an

amino group to give aspartic acid, or an acetic acid to give citric acid and so

on.

Enzymatic reactions of oxaloacetic acid

Each of the reactions of oxaloacetic acid is catalysed by its own

separate enzyme which catalyses only that reaction and none of the others.

Partly because of this specificity and partly because they take place at

relatively low temperatures, enzyme-catalysed reactions are much more

nearly quantitative that the analogous chemical reactions brought about in

laboratory by other agents.

(c) Substrate specificity: The extent of substrate specificity varies from

enzyme to enzyme. A few hydrolases are relatively nonspecific, others

356

require substrates containing certain groups (group specificity, e.g. -

galactosidase and -glucosidase, which cleave all galactosides and

.glucosides, respectively), while still other enzymes react very specifically

with one substrate only viz. urease which catalyses the hydrolysis of urea.

NH3CONH2 O

2H

2NH3 + CO2

Other important examples of group specificity are trypsin and

chymotryspin. Trypsin hydrolyses residues only of lysine and argenine,

while chymotrypsin hydrolyses residues only of aromatic amino acids (Phe,

Tyr and Try).

The study of the effects of such highly specific enzymes can give

useful information about the arrangement of amino acid residues in a

protein.

Similarly, invertase is a hydrolyzing enzyme which hydrolyses the

several fructosides, viz. sucrose and methyl fructoside.

Glucose - O - Fructose Invertase Glucose + Fructose

Sucrose H2O

H3C - O - Fructose Invertase CH3OH + Fructose

Methyl fructoside H2O

357

Check your Progress - 1

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) Enzymes are complex................molecules present in............

where they act as...................and bring about changes

in..............The word enzyme was coined by................in...........

from the.............term, meaning.

(ii) The enzymes are classified according to the general...................

they catalyze. There are six important classes:

(a) ............................ (b) ............................

(c) ............................ (d) ............................

(e) ............................ (f) ............................

(iii)Various micro organisms like................, ............ and .............

produce different kinds of.....................

(b) (i) For purification of enzymes the methods used are:

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(ii) Enzymes have.................., ................... macro molecules. The

molecular weight of enzymes various in between....................

(iii) The two most important general properties of enzymes are:

(a)

358

(b)

7.7 MECHNISM OF ENZYME ACTIVITY

According to most acceptable hypothesis the enzyme combines with

the substrate to form an intermediate, enzyme-substrate complex, which then

breaks down into product [P] and enzyme back. The latter enzyme can again

combine with the fresh molecule of the substrate in a similar manner. The

formation of enzyme substrate complex as an intermediate during the

reaction has been proved by spectroscopic studies.

E + S ES E + P

In the formation of enzyme-substrate complexes, the substrate

molecules attach at certain specific points on the enzyme molecule. These

specific point on enzyme molecules where the substrate molecules attach are

known as active, substrate or catalytic sites. Active sites on the enzymes are

usually provided be free hydroxyl group of serine (as in trypsing,

chymotrpsin, thrombin, alkaline phosphatase, cholinesterase and elastase),

phenolic group of tyrosine (as in pepsin), sulphydryl group of cysteine, or

imidazolyl group of histidine.

7.7.1 Fischer's Lock and Key hypothesis

Initially, Fischer in 1984 proposed that the substrate fits into the active

centre of the enzymes as a key fits into the lock. This lock and key theory for

enzyme action may be shown diagramatically in Fig. 7.2

359

Fig. 7.2

Thus according to the lock-and-key theory there are exact functional

groups and structural features in the enzyme into which the substrate

molecule must fit. The region of the enzyme that complexes with the

substrate; called the active site. The theory is somewhat restrictive in that it

allows very little variation in substrate dimensions. These are instances

where although a substrate may complex through the lock-and-key

mechanism with an enzyme, no reaction ensures. Moreover, in certain cases

a catalytic activity is observed even though a 'fit' is impossible.

7.7.2 Koshland's Induced fit Hypethesis

In order to account for the above observations, the Fischer's lock-key

mechanism was modified by Koshland in 1963, in the form of induced fit

mechanism. The essential feature of the Koshland's induced fit model is the

flexibility of the region of the active site. In order words, we can say that in

the Fischer model the active site is presumed to be pre-shaped to fit the

substrate, while in the induced fit model the substrate induces a

conformational change in the enzyme so that the substrate fits the active site

in the most convenient way. This explains why the enzymes become inactive

in denaturation because the latter phenomenon destroys the tertiary and

quaternary structure of the enzyme and thus the substrate can't induce

conformation changes in the enzyme to fit the substrate on the active sites.

360

Fig. 7.3: Representation of an induced fit by a conformational change in

the enzyme structure.

The active sites on the enzyme molecule exert a binding force on the

substrate molecule by hydrophilic as well as hydrophobic groups. Enzyme-

substrate complexes are formed by multiple bonding (covalent, hydrogen or

electrostatic) with the substrate.

The functional groups of the active sites are arranged, in a definite

spatial manner and thus only those compounds which can fit into this

definite spatial manner very well can function as the substrate of the

enzyme. This explains why only the d-isomer of a single substance can act

as substrate for a particular enzyme.

The enzymes requiring coenzymes for their activity also possess site

for attachment of coenzyme. The complex formed in such cases are known

as enzyme-substrate-coenzyme complexes.

7.7.3 Identification of Active Sites: Use of Inhibiters

Enzyme inhibitors are finding more and more importance in the basic

sciences as well as in the clinical field. Certain enzyme inhibitors

particularly the competitive type function as drugs, hence they are used to

explain the action of drugs and in ascertaining the active sites on the enzyme

molecules. The first notable example where the sulphonamides which are

found to be the competitive inhibitors of p-amino benzoic acid (a bacterial

361

growth factor). p-Aminobenzoic acid forms a part of an essential coenzyme,

called tetrafolic acid, made by the bacterial cell.

COOH SO2NH2

p-amino benzoic acid p-Aminobenzene sulphonamide

(Sulphanilamide)

The sulphonamide, being similar in structure, competitively inhibit the

utilization of the p-amino benzoic acid by the microorganism with the result

folic acid production in the micro-organism is prevented and thus growth of

the micro-organism stops. It is important to note that as the host obtains its

folic acid requirement from the diet and does not a synthesize it from the p-

amino benzoic acid, it remains unaffected as far as folic acid requirement is

concerned. Hence sulphonamides can be toxic to micro-organisms but

virtually harmless to the host.

Competitive inhibition also explains the action of several of the drugs.

For example, ephedrine and empheatmine prolong effects of the hormones

like adrenaline, noradrenaline and 5-hydroxytryptamine by competitively

inhibiting the enzyme monoamine oxidase (MAO) which brings about the

oxidative deamination of the above mentioned hormones.

362

A similar explanation has been put forward for the action of cocaine

and of ant depressive drugs like iproniazid and tranylcypromine.

The ability of some antideopessive drugs to inhibit monoamine

oxidase may also have serious side effects. Monoamine oxidase normally

brings about the oxidation of tvramine (formed by the decarboxylation of

tyrosine), but in presence of MAO inhibitors this may not happen and

tyramine may thus enter the general circulation and releases noradrenalin

from the stores. The inhibitor also prolongs the action of noradrenalin and

the end-result may be a very serious rise in blood pressure.

Another example of the drug whose function depends upon its

competitive inhibiting nature is allopurinol. The latter is very similar in

structure to hypoxanthine and thus inhibits the enzyme xanthine oxidase

363

in bringing about the oxidation of hypoxanthine and xanthine to uric acid

and is thus used in the treatment of gout a disease in which excess of uric

acid is deposited.

Enzyme Modification: Site Directed Mutagenenesis

Genetic engineering for industrial production of enzyme is possible by

transfer of genes encoding useful enzymes into a suitable host

microorganism (fig. 7.4). Introducing more copies of gene into the

concerned organism may increase the level of production of an enzyme. A

recent example of this technology is the detergent enzyme lipolase produced

by Novo Nordisk A/S, which has improved removal of fat stains in fabrics.

The enzyme was first identified in the fungus. Humicola languinosa. The

DNA fragment for the enzyme was cloned into production fungus

Aspergillus oryzae and commercial levels of enzyme production achieved.

The enzyme is very stable at a variety of temperature and pH condition

relevant to washing. In addition, lipolase is remarkably resistant to

proteolysis activity of the commonly used detergent proteases.

The modification in the catalytic activity for the usefulness of the

existing enzyme or production of new enzyme activity by making suitable

changes in its amino acid sequence is known enzyme engineering. Since all

enzymes are proteins, enzyme is an integral part of protein engineering. The

main objective of the approach is to modify various properties of the

enzyme, so that enzyme become more useful.

Cultivation of microgranism

with useful enzyme

364

Isolation and Purification Isolation and Purification

of enzyme of m RNA

Determination of Partial m RNA verseRe DNA

transcriptase

Synthesis of Cloning of DNA in

oligonucleotide probe Ecoli. (C DNA

Identification of CDNA Clone by

suitable hydridisation technique

Transforming Industrial host

organism. Aspergillus Orzyae

Industrial Production of

desired enzyme

Fig. 7.4 Genetic Engineering for

Industrial Enzyme Production

The main objective of enzyme engineering is to produce an enzyme

which is more useful in industrial and of the applications by modifying them

in order to:

improve the activity of enzyme

enhance the stability

alter optimal pH and temperature

increase thermostability

365

after the specificity of an enzyme so that it catalyses the conversion of

different substrate

improve the efficiency of a process

The alternation in the properties of an enzyme is always reflected in

its primary structure i.e. the amino acid sequence. Protein engineering is

rightly called molecular surgery as it introduces the amino acid changes in

certain critical regions of the protein. The protein engineering of enzyme in

achieved by developing a three-dimensional graphical model of purified

enzyme with the help of x-ray crystallography, nuclear magnetic resonance

(NMR), etc.. Using this data, a molecular model is prepared for determining

a possible change in the sequence of enzyme. This can be achieved by two

methods. The first method is the mutagenesis of cloned-gene product.

Amino acid residues at defined position in the structure of enzyme can be

replaced by other suitably coded amino acid residues. The altered gene is

then introduced and expressed in a suitably coded amino acid residues. The

altered gene is then introduced and expressed in a suitable host i.e. E.coli

and the mutant enzyme subsequently produced with the requisite changes in

position. This approach is called site-directed mutagenesis. The second

method involves the isolation of the natural enzyme and further modification

to its structure can be carried out by chemical or enzyme treatment.

Sometimes this approach is called chemical mutation.

The enzyme subtilisin (obtained from Bacillus amyloliquefaciens)

used in detergents and the lactate dehydrogenase (isolated from Bacillus

stearothermophilus) are the examples of enzyme engineering by which the

amino acid is replaced to alter their performance. In addition the enzyme

phospholipase A, which is used as a food emulsifier, was modified

366

structurally to resist higher concentrations of acid. Genetic engineering and

protein engineering will have a significant role to play in the enzyme

industry, since they ensure better product economy, production of enzymes

from rare microorganisms, faster development programmes and above all,

such enzymes are ecofriendly and have low allergenic potential.

7.8 ENZYME KINETICS

As has been pointed out (section 7.4) rate of enzyme reaction depends

on various factors, mainly temperature, pH and substrate concentration.

Kinetic studies by Michaelis-Menten and lineweaves-Burk, involving double

reciprocal plot indicate formation of enzyme-substrate complex.

7.8.1 MICHAELIS-MENTEN PLOT

It was Michaelis and Menten in 1913 who proposed a successful

explanation for this observation. According to them the enzyme and the

substrate S combine rapidly to form a complex, the enzyme-substrate

complex, ES. This complex then breaks down relatively slowly to form the

product P of the reaction.

E + S

2

1

K

K

ES

E S 3K E + P

Applying law of mass action to the first step of the reaction.

The rate of forward reaction = K1 [E] [S]

The rate of backward reaction = K2 [ES]

Since at equilibrium the rates of the two reactions are equal,

K1[E][S] = K2 [ES] ........(1)

Divide both the sides by K1 [ES]

367

}[

}[

][

][][

1

2

1

1

ESK

ESK

ESK

SEK

mKK

K

ES

SE

1

2

][

][][ ........(2)

where Km is the Michaelis constant.

Now, if [E0] is the concentration of the total enzyme, then at any time

[E] + [ES] = [Eo]

Subtract [ES] from both sides of the above equation

[E] = [Bo] - [ES]

Substitute the value of [E] in equation (2)

]ES[

}s{}]ES[]E{[K 0

m

Multiply both the sides by [ES]

[Km] [ES] = [Eo] [S] - [ES] [S]

or [ES] {Km + [S]} = [Eo] [S]

Divide both the sides by {Km+ [S]}

][

][][][

SK

SEES

m

o

........(3)

Now according to Michaelis and Menten, the slowest (rate

determining) step of the, reaction is the breakdown of the enzyme substrate

complex, ES. Thus the velocity v of the overall reaction, according to Law

of Mass action, is given by

v = K3[ES]

Substitute the value of [ES] from equation (3)

368

]S[K

]S[]E[Kv

m

o3

........(4)

Now if the enzymes are completely saturated with the substrate, v will

increase to the maximum velocity V which according to Law of Mass action

will be given by

V = K3 [Eo]

Substitute the value of K3 [Eo] in equation (4)

][

}[

SK

SVv

m ........(5)

This is the Michaelis-Menten equation. Now when v is equal to half

of the maximum velocity (V), i.e.

2

Vv

then ][

][

2 SK

SVV

m

or 2[S] = Km + [S]

[S] = Km

Thus the substrate concentration, [S], required for half

369

Concentration of substrate [S]

Fig. 7.5: Michaelis plot showing determination of Michaelis constant, Km

Attainment of the maximum activity of an enzyme is known as

Michaelis constant*. Thus Michaelis constant may be determined from a

plot, commonly known as Michaelis plot, obtained by plotting substrate

concentration, [S] versus rate of reaction, v (Fig. 7.5).

Michaelis constant is a characteristic of an enzyme, at definite pH

and temperature. Typically the values of Km lie in the range 10-2

to 10-5

moles/litre for various enzymes. Km value is very useful in evaluating

affinity of the enzyme for a substance; the low Km value of an enzyme

indicates its high affinity and vice versa. The Km value also gives an idea

regarding the type of inhibition of an enzyme caused by an inhibitor.

7.8.2 Line Weaver Plot

The value of Km may be obtained more accurately from the

Lineweaver-Burk equation which is obtained by inverting the Michaelis-

Menton equation, (5). ,

][

][

SV

SK

v

I m

][SV

K

V

I

v

I m ........(6)

This is known as Lineweaver-Burk equation. It is used for

determining the Michaelis constant for which the reciprocals of the observed

370

velocities (I/v) are plotted against the reciprocals of the corresponding

substrate concentrations (1/S) keeping, the amount constant (Fig. 7.6).

Fig. 7.6: Lineweaver-Burk plot to determine Km

A linear curve is obtained which when extrapolated intercepts the I/S

line which gives the value of -1/Km from which Km can easily be calculated.

Actually, the substrate binds to the active site of the enzyme molecule

and as the substrate is added more active sites of the enzyme molecule take

part with the result the reaction velocity increases. But since the number of

active sites on an enzyme molecule are fixed a stage will come when whole

of them have combined with the substrate molecules (saturation of the

enzyme) at which stage the enzyme will be working at its maximum

velocity. Now since none of the active sites of the enzyme is free, further

addition of the substrate molecule will not further increase reaction velocity.

371

Fig. 7.7: Graph of reaction velocity against increasing substrate

concentration at constant enzyme concentration.

The velocity of a catalysed reaction is proportional to the

concentration of catalyst. In case the enzyme concentration [E], is doubled,

then as much as twice active sites become available to combine with

substrate provided an excess of substrate is present, and thus the maximum

velocity is also doubled. Therefore, in general v [E]. This relationship is

illustrated in Fig. 7.8.

Fig. 7.8: Reversible and Irreversible Inhibition

372

7.8.3 Reversible and Irreversible Inhibition

Since enzymes are proteins, any agent which denatures proteins will

inactivate enzymes. Such agents are known as inhibitors of enzymes and

thus may be defined as the chemical substances that reduce the activity (i.e.

velocity) of particular enzyme. They may be small inorganic ions such as

cyanide, which inhibits the enzyme called cytochrome oxidase, or much

more complex inorganic or organic molecules. This phenomenon in which

the enzyme activity is decreased by the presence of inhibitors is known as

inhibition. It may be of two types:

(a) Reversible Inhibition

(b) Irreversible Inhibition

Reversible Inhibition

In this type of inhibition there is non-covalent bonding between

inhibitor and enzyme. In this type of inhibition there are many modes. These

modes depend on the fact that how and by what mechanism the mixing of

inhibitor decreases the activity of enzyme or how it affects kinetics of the

reaction.

Reversible inhibitors easily associate and dissociate with the enzyme.

When they are bound with the enzyme, they make them active:

E + I EI

where I = Inhibitor.

Like ES complex, in EI complex also E is linked with I, using weak

non-covalent interaction. Reversible inhibition can be divided in to:

1. Competitive inhibition: This type of inhibition occurs when the so-called

inhibitor competes with the proper substrate for binding at the active site of

373

the enzyme. In such type of inhibitions, both enzyme-substrate (ES), and

enzyme-inhibitor (El) complexes are formed during the reaction. The

relative amounts of the two complexes depend partly upon the affinity of the

enzyme towards the substrate and inhibitor and partly upon the relative

concentration of substrate and the inhibitor. Thus if the inhibitor is present in

sufficiently high concentration, it can displace the substrate entirely and thus

blocks the reaction. On the other hand, the degree (i.e. percentage) of

inhibition observed for a competitive inhibitor at a particular concentration

can be reduced by increasing the substrate concentration (see fig. 7.9).

Substrate concentration

Fig. 7.9: Michaelis plot for a competitive inhibitor

This competition also follows the law of mass action. The extent of

inhibitor binding may be expressed by the inhibitor constant (analogy to the

substrate constant, Ks).

]EI[

]I[[E[K

s

It is observed that the competitive inhibitor has similar size and

structure to the corresponding substrate which is evident from the tables of

374

some important competitive inhibitors of the corresponding enzyme-

substrates.

2. Non-competitive Inhibition : Many enzymes can be poisoned more

or less specifically by different substances, i.e. they are completely

inactivated. The phenomenon is known as non-competitive inhibition and

the reagents which bring about this effect are known as non-competitive

inhibitors. These compounds generally bear no structural similarity to the

substrate and unlike competitive inhibition, the process is usually

irreversible. Such compounds are often reagents, which are capable of

reacting covalently with functional groups (viz. - OH, - SH, NH2 and > NH)

of the active site of the enzyme and thus make the latter inactive for the

substrate. A good example of this type is the irreversible inactivation of the

so-called SH enzymes by the reaction with indoacetamide or N-

ethylmaleimide. Reaction of diisopropylfluorophosphate with a hydroxyl

group of the serine residue in the enzyme, acetylcholine esterase, results in

complete loss of activity of the enzyme. Thus diisopropylflu-orophosphates

and similar phosphate and thiophosphate esters act as never poisons by

inhibiting acetylcholine esterase. This type of inhibition may also occur

when anions interact with a cation which is associated with the active site,

e.g. anions like 2CN

-, F

-, S

--, and oxalate inhibit enzymes containing the

cations like Fe+3

, Mg+2

, Cu+2

, and Ca+2

, respectively. This type of inhibition

is overcome by the additions of powerful chelating agents, viz. EDTA,

which form complexes with the metal ions.

It is important to note that non-competitive inhibitions is not always

irreversible, it may also be reversible.

375

Lastly, unlike the competitive inhibition, in non-competitive

inhibition since no competition with substrate is occurring, the percentage

inhibition is not reduced by increasing the substrate concentration, i.e. the

extent of inhibition is independent of the concentration of the substrate.

3. Allosteric inhibition (end product inhibition): In this type of inhibition,

the enzyme has two different sites; viz, the active site, where the inhibitor

may bind. The inhibitor present at the allosteric site may affect the

conformation at the active site with the result it becomes difficult for the

enzyme to take the substrate molecule and, in the extreme case, the enzyme

completely fails to take up the substrate molecule. Allosteric inhibition is of

great physiological significance.

For illustrating consider the substrate L-threonine which is converted

into L-isoleucine (end-product) through various intermediate. Now if the end

product combines with the active site of the enzyme, the inhibition is of the

competitive type. Although this does occur , more frequently the end product

combines with the allosteric site on the enzyme altering the conformation of

the latter and thus inhibits attack of substrate on the enzyme and thus

ultimately the reaction. Thus the accumulation of the end product slows

down the whole reaction sequence by inhibiting an earlier step and finally

brings it to a halt.

Now as the end product (which is acting as an inhibitor) is consumed

synthesis resumes because allosteric inhibition is reversible.

Irreversible Inhibition

Some substances combine with enzymes with covalent bond and

make enzyme inactive irreversibly. Almost all crreversible enzyme

inhibitors, natural or synthetic are poisonous substances. In most of the cases

376

these substance, block the active sites of enzymes for substrate, by

combining with the functional groups present there i.e. they are not available

for substrates. These substances are generally -OH, -SH, -NH2 or NH

groups.

An important example of irreversible inhibitor is iodoacetamide or N-

ethyl melemide, the -SH group of the inhibitor combines with free enzyme

and reduces its reactivity. Diisopropylfluorophosphates (DFP), combines

rapidly with -OH group of amino acid serine irreversibly to give additive

compound. The bond is covalent.

Thus DFP functions as irreversible inhibitor for such enzymes which

have serine in their active centers.

O

||

E CH2-OH + (CH3)2 CHO P OCH (CH3)2

| O

F ||

DFP (CH3)2 CHOPO CH (CH3)2+HF

|

OCH2-E

Enzyme acetylcoline esterase has this type of arrangement. Acetyl

coline is a neurotransmitter which works in certain specific portions of

neuron tissue. Stimulate nerve cell releases acetocoline in synapse. The free

acetocoline combines with receptor site of second neurocell and thus

transmits nerve impulse. Before the second impulse is transmitted, the

acetocoline released by the first impulse must be hydrolised into acetate and

coline. This hydrolysis takes place in presence of acetycoline esterase. These

hydrolysis products have no ability for transmission. DFP makes the enzyme

inactive by combing with -OH group of serine residue. This inhibition

377

readily paralyse vital functions. Many pesticides have structure similar to

DFP, which act as strong inhibitor for acetylcoline esterase.

Many natural poisonous substances are irreversible enzyme inhibitors.

The poisonous substance present in Calabar beans is an alkaloid

physiostigmin, which acts as a potent in hibitor for acetylcoline esterase

enzyme.

Some irreversible inhibitors are very selective, because they resemble

with the substrate, rather than with the intermediate state. For example, N-

tossylphenyl alanine chloromethyl Ketone (TPCL) is a good inhibitor for

Kimotrypsin.

O

||

C6H5CH2 ― CH ― C ― CH2Cl

|

NH

|

O = S ― C6H4CH3

||

O

Its phenyl group comfortably fit into the active centre of the enzyme

and chlorine atom is arranged in such a way that nucleophilic substitution

may takes place with nitrogen of emidezol ring of His 57 enzyme. Many

compounds of the type TPCK have been synthesized for inhibition of

enzyme reactivity.

In addition, organophosphorous, organomercury compounds, cyanide,

carbon monoxide, hydrogen sulphide etc. also act as irreversible inhibitors.

Check your Progress - 2

Notes :(i) Write your answer in the space given below .

378

(ii) Compare your answer with those given at the end of the unit.

(a) (i) Enzymes combines with……………..............…to form an

intermediate……............ which then breaks down into…......… and

.................. back.

(ii) Fischer proposed that the…….…….fits into the……………..of

the…………….as a key fits into the……….. while…………

modified this mechanism by its…………..

(iii)Active sites of an……....…..is ascertained by using….......…..

Protein engineering is called……….as it introduces…… in

certain critical regions of the…………. . This approach is

called……………

(b) (i) Kinetic studies by……………. and…………… involving……

plot indicate formation of a………………

(ii) The chemical substances that…………….. the……………..

of………….. are known as………….Reversible inhibition are of

there types:

(a) .................................................

(b) .................................................

(c) .................................................

(iii)Almost all irreversible enzyme inhibitors are……………they

block the……………of………………..for………….

7.9 LET US SUM UP

By going through this unit you would have achieved the objectives

state at the start of the unit. Let us recall what we have discussed so for:

379

Enzymes are complex protein molecules present in living cell which

catalyze and control all biochemical reactions in every living things.

Every cell synthesize its own enzymes and a single body cell contains

as many as 100000 enzymes, each directing a specific reaction, each

coming into play at the right moment and place.

Most of the enzymes, produced by a cell, function within that cell and

hence are called endo-enzymes but some enzymes are liberated by

living cells and catalyse reactions in the cell's environment, such

enzymes are known as exoenzymes.

W.Kuhne coined the work 'enzyme' in 1878 from Greek term meaning

yeast.

On the basis of the type of reactions that they catalyse, enzymes are

classified into six main classes. They are named by adding suffix 'ase'

to the name of the substrate, e.g. oxidoreduetase, transferase,

hydrolase, lyase, isomerase and ligase.

Unlike the catalysts employed in the chemical laboratory which can

withstand quite several conditions, the activity of enzymes is

markedly affected by several factors viz. temperature, pH and

concentration of other ions. They exhibit highest activity under

optimal conditions.

Various microorganisms like bacteria, fungi and yeast produce

different kinds of enzymes, even the animal tissues and plants are

used as sources of enzymes.

For isolation of intracellular enzymes from a cell one of the following

processes may be used:

380

(i) Crushing the tissue with sand,

(ii) Crushing the tissue in a homogenizer,

(iii) By crushing the tissue with acetone, lipid of the cell

dissolves in acetone and get separated. The enzymes is

obtained as the residue.

The purification of the enzymes is done using:

(i) Solvent extraction

(ii) Temperature and pH denaturation

(iii) Differential denaturation

(iv) Electrophoresis

(v) Selective adsorption and elution of proteins

(vi) Use of sephedex molecular sieve

(vii) Column Chromatography

Enzymes are soluble and colloidal proteins and have large

macromolecules with molecular weights generally in between 12000-

10000.

Due to large size their rate of diffusion is very low. The two general

properties of enzymes are their tremendous efficiency and their

remarkable specificity.

According to most acceptable hypothesis the enzyme combines with

substrate to form an intermediate enzyme-substrate complex, which

then breaks down into product and enzymes back:

E + S ES E + P

381

In the formation of enzyme-substrate complexes the substrate

molecules attach at certain specific points on the enzyme molecules,

called active centre or active site.

Fischer proposed (1984) that the subtrate fits into the active centre of

the enzyme as a key fits into the lock. According to the lock and key

theory there are exact functional groups and structural features in the

enzyme into which the substrate molecule must fit.

Fischer's lock and key mechanism was modified by Koshland in 1963,

in the form of induced fit mechanism.

In the Fischer's model the active site is presumed to be pre shaped to

fit the substrate, while in the induced fit model the substrate induces a

conformational change in the enzyme so that the substrate and active

site come to each other in such a way that the substrate fits the active

site in the most convenient way.

The competitive type enzyme inhibition is used to explain the action

of the drug and to ascertain the active site on the enzyme molecules.

The main objective of enzyme engineering (transfer of gene's

encoding useful enzymes into a suitable host micro organism) is to

modify various properties of the enzyme, so that the enzyme become

more useful.

The rate of enzyme-reaction depends on various factors, mainly

temperature, pH and substrate concentration. Kinetic studies by

Michaelis-Menten and Lineweaver-Burk involving double reciprocal

plot indicate formation of enzyme substrate complex.

382

Enzyme inhibitors are the chemical substances that reduce the activity

of particular enzyme. The inhibition process may be either (i)

Reversible or (ii) Irreversible.

In reversible inhibition there is non-covalent bonding between

inhibitor and enzyme and may be of three types: (i) Competitive

inhibition, (ii) Non-competitive inhibition and (iii) Allosteric

inhibition.

Irreversible inhibitors are generally poisonous substances, which

combine with enzymes with covalent bond and make enzyme inactive

irreversibly. In most of the cases they block the active sites of

enzymes for substrate, by combining with the functional groups

present there.

7.10 CHECK YOUR PROGRESS: THE KEY

1 (a) (i) protein

living cells

bidogical catalysts

substances

W.Kuhne

1878

Greek

yeast

(ii) type of chemical reactions

(a) Oxido reeducates (d) Lysases

(b) Transferases (e) Isomerases

(c) Hydrolases (f) Ligases

383

(iii) bacteria

fungi

yeast

enzymes

(b) (i) (a) Precipitation

(b) Solvent extraction

(c) Temperature and pH denaturation

(d) Differential Centrifugation

(e) Electrophoresis

(f) Selective adsorption

(g) Sephedex molecular seiving

(h) Column Chromate graphy

(ii) Soluble

Colloidal

12000-100000

(iii) (a) Tremendous efficiency

(b) Remarkable specificity

2 (a) (i) substrate

enzyme-substrate complex

product

enzyme

(ii) Substrate

active centre

enzyme

lock

Koshland

induced fit mechanism

384

(iii) enzyme molecule

competitive type inhibitor

molecular surgery

amino acid changes

protein

(b) (i) Michaelis-Menten

Line weaver-Burk

double reciprocal

enzyme-substrate complex

(ii) reduce

activity

particular enzyme

inhibitors

(a) Cp,[etotove omjobotopm

(b) Non-competitive inhibition

(c) Allosteric inhibition

(iii) poisonous substances

active sites

enzymes

substrates.

385

UNIT-8 ENZYME ACTION

Structure

8.1 Introduction

8.2 Objectives

8.3 Mechanism of Enzyme Action.

8.3.1 Transition State Theory.

8.3.2 Orientation and Steric Effect.

8.3.3 Modes of Enhancement of Rates of Bond - Cleavage:

(a) Acid-Base Catalysis.

(b) Covalent Catalysis.

(c) Strain or Distortion,

8.4 Examples of Some Typical Enzyme Mechanisms.

8.4.1 Chymotripsin.

8.4.2 Ribonuclease.

8.4.3 Lysoenzyme.

8.4.4 Carboxypeptidase-A

8.5 Kinds of Reactions Catalysed by Enzymes:

8.5.1 Nucleophilic Displacement on a Phosphorus.

8.5.2 Multiple Displacement Reactions and the Coupling of ATP

Cleavage to Endergonic Process.

8.5.3 Addition and Elimination Reaction.

8.5.4 Enolic Intermediates Isomerisation Reactions: Po cleavage and

Condensation.

8.5.5 Isomerisation and Rearrangement Reactions.

8.5.6 Enzyme Catalyzed Carboxylation and Decarboxylation.

386

8.6 Let Us Sum Up

8.7 Check Your Progress: The Key

387

8.1 INTRODUCTION

We know enzymes are important group of bimolecules synthesized by

the living cells. They are catalysts of biological systems (hence are called as

biocatalysts), colloidal, thermolabile and protein in nature. They are

remarkable molecular devices that determine the pattern of chemical

transformations. They also mediate the transformation of different forms of

energy. The striking characteristics of enzymes are their catalytic power and

specificity. Actions of most enzymes are under strict regulation in a variety

of ways. Substances on which enzymes act to convert them into products are

called substrates.

As has been pointed out enzymes have immense catalytic power and

accelerate reactions at least a million times, by reducing the energy of

activation. Before a chemical reaction can occur, the reacting molecules

required gaining a minimum amount of energy, this is the energy of

activation. It can be decreased by increasing the temperature of the reaction

medium. But in human body which maintains a normal body temperature

fairly constant, it is achieved by enzymes. In general we can say practically

all biochemical reactions are catalyzed by one or more enzyme system.

It is will known in general with the exception of ribozymes which are

few RNA molecules with enzymatic activity, "all the enzymes are protein in

nature with large mol. wet". Few enzymes are simple proteins while some

are conjugated proteins. In such enzymes the non-protein part is called

prosthetic group or coenzyme and the protein part is called as apo-enzyme

The complete structure of apoenzyme and prosthetic group is called as

holoezyme :

Holoenzyme = Apoenzyme + Coenzyme.

388

Certain enzymes with only one polypeptide chain in their structure are

called as monomeric enzymes, e.g. ribonuclease. Several enzymes possess

more than one polypeptide chain and are called as oligomeric enzymes, e.g.

lactate dehydrogenase, hexokinase, etc. Each single polypeptide chain of

oligomeric enzymes is called as subunit. When many different enzyme

catalyzing reaction sites are located at different sites of the same

macromolecule, it is called as 'multienzyme complex. The complex

becomes inactive when it is fractionated into smaller units each bearing

individual enzyme activity, e.g., fatty acid synthesize, carbamoyl phosphate

synthetase II, pyruvate dehydrogenase, prostaglandin synthase etc.

Michaelis and Menten have proposed a hypothesis for enzyme action,

which is most acceptable. According to their hypothesis, the enzyme

molecule (E) first combines with a substrate molecule (S) to form an

enzyme-substrate (E S) complex, which further dissociates to form product

(P) and enzyme (E) back. Enzyme once dissociated from the complex is free

to combine with another molecule of substrate and form product in a similar

way.

The ES complex is an intermediate or transient complex and the

bonds involved are weak non-covalent bonds, such as H-bonds, Van der

Waal's forces hydrophobic interactions. Sometimes two substrates can bind

to an enzyme molecule and such reactions are called as bisubstrate

reactions. The site to which a substrate can bind to the enzyme molecule is

extremely specific and is called as active site or catalytic site. Normally the

molecular size and shape of the substrate molecule is extremely small

compared to that of an enzyme molecule. The active site is made up of

several amino acid residues that come together as a result of folding of

389

secondary and tertiary structures of the enzyme. So, the active site possesses

a complex three dimensional form and shape, provides a predominantly non-

polar cleft or crevice to accept and bind the substrate. Few groups of active

site amino acids are bound to substrate while few groups bring about change

in the substrate molecule.

8.2 OBJECTIVES

The main aim of this unit is to explain mechanism of enzyme action.

After going through this unit your would be able to:

discuss different theories of enzyme activity.

explain the activities of some typical enzymes and

understand different kinds of reactions catalyzed by enzymes.

8.3 MECHANISM OF ENZYME ACTION

According to Michaelis Menten hypothesis enzyme molecule (E) first

combines with a substrate molecule (S) to from an enzyme-substrate

complex (ES) which further dissociates to form product (P) and enzyme (E)

back.

Thus, enzymes are biochemical catalysts involved in the chemical

transformation of a specific compound or a group of compounds (the

substrates). It has been established enzyme activities depend upon the

presence of various metals. The metal, in an enzyme, is present as a chelate.

On the basis of the ease with which the metal can be removed from the

enzymes, Vallee designated" them as (i) metallo-enzymes (inert complexes)

in which the metal does not leave the enzyme easily, even in the presence of

large amounts of a good coordinating agent, and (ii) metal enzyme

complexes (labile complexes), from which the metal can be removed easily.

390

The former involves a specific metal and analogous complexes with

different central metal ions having similar charge and size, which do not

behave catalytically in the same manner. In the metal enzyme complexes,

the metal requirement is usually of a more general nature and may be

satisfied by a range of ions having similar charge and size; thus Mg(II) and

Mn(II) are often both effective as activators of many enzymes. The metal

ions, which are found in various enzyme systems, are: iron, copper,

manganese, zinc. magnesium, molybdenum and calcium.

8.3.1 Transition State Theory

Formation of enzyme substrate intermediate complex during enzyme

activity in physiological system is very complex, and the precise mechanism

of these interactions is almost unknown. It is reported that the activity is a

combination of steric, electronic and pharmokinetic factors and it could be

understood in the light of chelating theory i.e. participation of specific metal

ions during intermediate complex formation. Irrespective of whether a metal

ion forms the integral part of the active site of an enzyme, or, the enzyme

requires the metal ion as a cofactor for its activity, mixed ligand complexes

involving metal ions, enzymes and the substrates are formed in most

enzymic reactions. The specific role played by a metal ion in an enzymic

reaction is often difficult to elucidate due to the complex nature of the

protein structure. Model studies with relatively simple molecules of known

structures often yield valuable information that gives clues to the roles of

metal ions in many enzymic reactions. Metals are supposed to serve two

purposes: (i) provide proper stereo-chemical orientation and (ii) bring

reacting molecules (the enzyme and substrate) closer so that the reaction

may occur.

391

Three probable mechanisms have been proposed to explain the mode

of action of enzymes:

(1) The metal ion, M. forms complex with protein part of the enzyme, E

(apoenzyme), forming the active enzyme, EM (Eq.l). The active enzyme

reacts with the substrate to give an intermediate,

E + M EM .....................(1)

enzyme-metal-substrate complex (EMS), which subsequently

decomposes into products, P, (derived from the substrate, S), and the active

enzyme :

EM+S EMS .......................(2)

EMS EM + P .......................(3)

(2) The metal ion forms complex with the substrate first. The metal-substrate

complex (MS) reacts with the enzyme to give the enzyme-metal-substrate

complex (EMS), which subsequently decomposes into the products, the

enzyme and the metal, i.e.

M+S MS .......................(4)

E + MS EMS .......................(5)

EMS P + E + M .......................(6)

(3) The enzyme reacts with the substrate to form enzyme-substrate complex

(ES), which then combines with the metal ion to give enzyme-substrate-

metal complex (ESM). This (ESM) decomposes subsequently to furnish the

products:

E + S ES .......................(7)

ES + M EMS .......................(8)

ESM P + E + M .......................(9)

392

Out of these three mechanisms, the first is followed by most of the

metal-enzyme complex systems. It finds support in the kinetic studies and in

the determination of equilibrium constants for the complexes involved. The

second mechanism, although less commonly reported, is followed in a

number of cases, viz. inorganic pyrophosphatase, hexokinase and creatine

phosphokinases6.

The enzyme is often inactive towards simple derivatives of these

substrates. In many cases, the simple replacement of hydrogen atom by a

methyl group completely blocks the reaction. The elucidation of the

chemical mechanism by which enzymes catalyze reactions is a difficult

problem. The most commonly used theory for providing a detailed

mechanism in these cases, is the polyaffinity theory of Bergmann, which

may be summed up as 'enzymes interact with multiple sites on the substrate'.

Calvin emphasized the suitability of the coordination process for

furnishing this second kind of action. Involvement of metal complexes in

enzymatic reactions was pointed out by Hellerman, while Smith postulated

the formation of mixed complexes as intermediates in metal activated

enzymatic reactions. Smith worked on leucine amino-peptidase and pointed

out that the enzyme functions through formation of Mn2+

or Mg2+

activated

complex, e.g. glycyl-L-leucinamide forms Mn(II) activated enzyme leucine

amino peptidase complex as shown in structure-1.

393

Orientation and Steric Effect

Smith listed the factors resulting from metal chelates, involved in

determining the specificity of these enzymes:

(a) The substrates should have specific polar groups and these must

combine with the enzyme during hydrolysis and presumed to form

chelates with the metal.

(b) The substrate must have the correct steric orientation, preferably the L-

orientation. The rate of the hydrolysis of L-leucine amide is about

1000 times faster than that of D-leucine amide.

(c) The substituents affect the energy of the peptide bonds. Thus, with

increase in the acid strength of the carboxyl group of the acid involve

in the peptide link, there is an increase in the rate of hydrolysis.

(d) The nature of the alkyl group attached to the -carbon atoms of the

peptide also affects the rate of hydrolysis.

It is believed that the biological response of an enzyme is the result of

its interaction with a receptor. Receptor may be considered as a locus on the

plasmic membrane with a structure which facilitates its preferential and

selective contact with enzyme molecule (at the surface or inside the cell

affected). Recent studies have shown them portentous in nature, usually a

membrane protein. They may be regarded as chemical groups on the

receptor (protoplasmic macromolecule) which give recognition site for

molecules having specific structure (the drug molecule), i.e. shape of the

enzyme molecule in three-dimensional space is crucial for union with the

receptor; molecules with a fixed conformation may only get fitted in these

sites. This suggests that the enzyme and their receptors share

complementary structure, which helps an enzyme to recognize its receptor

394

(so it is said, receptor response are genetically determined). This is

somewhat similar to the lock and key arrangement (i.e. enzyme-substrate

interaction). It is believed that metal ion helps in giving proper orientation of

the enzyme in the receptor site, thus reducing strain.

8.3.3 Modes of Enhancement of Rates of Bond - Cleavage

There is a group of enzymes which are involved in the natural process

of bond formation and bond-cleavage, such as-

(i) during cleavage of peptide bonds (i.e. metabolic decomposition of

protein into amino acids), the enzymes involved are endopeptidases, such as

chymotrypsin (Ca2+

-complex), exopeptidase (bivalent metal complexes),

dipeptidase such as glycylglycine dipeptidase (Co2+

-complex) or glycyl-L-

leucine dipeptidase (Zn2+

/Mn2+

-complex) and carboxypeptidases (Mg2+

-

complexs),

(ii) during carboxylation and decarboxylation reactions (i.e. the addition

and removal of carbon dioxide), such as conversion of oxalosuceinic acid to

-ketoglutaric acid and of -ketoglutaric acid to succinic acid, the

carboxylase enzymes involved are magnesium and manganese complexes,

while carbonic anhydrate is a Zn(II) complex,

(iii) during phosphorlation reactions (i.e. synthesis or destruction of

phosphate bonds, the energy source of biochemical reactions), such as

conversion of ATP to ADP by phosphorylase (Mg2+

-complex), or the

activity of contractile protein actomysoin (Mg2+

-complex) or the biological

activity of insulin (Zn2+

-complex), the metal-enzyme complexes function as

catalyst. Similarly, during the other condensation and cleavage reactions,

395

such as condensation of acetate in the form of acetyl coenzyme-A

(Mg2+

/Ca2+

-complex) with oxalio-acetic acid enol to form citric acid or the

function of enolase (Mg2+

-complex) during the dehydration of D-2,

phosphoglyceric acid to phosphoenol pyruvate depends on the metal-

enzyme-complex catalysis.

During these reactions many modes are involved which enhance rate

of bond cleavage e.g.

(a) Acid-base catalysis,

(b) Covalent catalysis, and

(c) Strain and molecular distortion

(a) Acid-Base Catalysis :

The reactions which are catalyzed by the presence of an acid or a base

in the reaction mixture are called 'Acid-Base Catalysis Reactions. These

reactions are divided into two groups: The general acid-base catalysis

reactions and the specific acid-base catalysis reactions.

The specific acid-base catalysis reactions are those reactions, the rates

of which are changed with the change in the concentration of H+ or H3O

+

ions in the solution; while they remain unaffected in the presence of other

acids or bases.

On the contrary, those reactions, whose rates are affected by the

presence of all acids and bases in the reaction solution, are known as

'General acid base catalysis' reactions. Rotation of glucose in a solution is a

general acid-base catalysis reaction.

Hydrolysis of an organic ester or amide is a specific acid or base

catalysis reaction. Thus, Acid-catalyzed hydrolysis of an amide may be

represented as follows:

396

O O—H O—H H H+ H +H2O H

R — C — N R — C— N R—C—N

R1 step I

R1

step II R

1

O—H O

H H+

R—C—N — H R— C — OH + R1NH2

step III

R1

step IV

O

H

The functional groups of the enzyme orient in the active centre in such

a way that they interact with the substrate effectively. In the substrate

proton-donar and proton-acceptor are located nearby. The functional groups

of amino acid in the active centre fulfill the function of acid-(-NH+

3 and

COOH) or base (NH2 and -COO-)

(b) Covalent Catalysis :

The side chains of amino acid consist of many nueleophilic groups for

catalysis, such as RCOO-, RNH2, aromatic -OH, histidene, R-OH and RS

-.

These groups attack on electrophilic part of the substrate and link the

substrate and enzyme by a covalent bond. This results in an intermediate

complex. The formation of this covalent bonded intermediate complex

results due to nucleophilic attack of the enzyme on substrate. Acylatation,

phosphorylation or glycolysation of nuclophile are examples of covalent

catalysis, e.g.

(a) Hydrolysis of Peptide Bond:

397

This reaction involves nucleophilic attacks of H2O on electron

deficient carbonyl carbon in the first step. Carbonyl group being an electron

attracting group, makes the carbon positively charged. In the second and

third steps iso tetrahedral intermediate compound is formed. In the last step

product is obtained as a result of rearrangement of electrons :

(b) Nucleophitic Attack of Enzyme on Phosphorus Atom :

This involves formation of an intermediate compound due to linking

of enzyme with phosphorus atom covalently :

R R R

En3—X: O R1 En3—X O R

1 En3—X O

O O

O = P O = P O = P+ - O ― R

O O O

The phenyl group of the substrate comfortably fits into the active

centre of the enzyme and the chlorine atom is oriented in such a way that

398

nucleophilic substitution becomes easy with nitrogen of Hi-57

imidezol ring

of the enzyme. Many compounds like TPCK have been synthesized to

explain mechanism of the enzyme.

(c) Strain and Molecular Distortion :

The strain present in reacting molecule is released in transition state

and the product is formed. In the following examples (a) and (b), the

reaction takes place 108 times faster in case of (a) compared to (b) :

The difference in the rates of these two reactions lies in the structures

of reacting molecules. In (a) since the reacting molecule is a cyclic

compound and has high bond-strain. The potential energy of (R) must also

be high because of its cyclic configuration. When (a) is hydrolyzed, open

chain compound is obtained as the product, which is free from the strain.

This effect helps in the increase of reaction rate. On the country the reactant

in (b) is an open chain compound and has no strain and which on hydrolysis

gives another open chain compound with no strain.

Similarly, it has been shown that the effect of strain becomes quite

evident when we compare enzyme catalyzed reaction with non-catalyzed

reactions :

399

(a) Uncatalysed reaction (b) Enzyme catalysed reaction

In unanalyzed reactions, the probability of formation of strained

configuration of reactions is less for the interaction of reacting groups.

While in enzyme catalyzed reactions. The substrate forms enzyme-substrate

complex on combination with the enzyme, the process the substrate has

tendency to go into transition state, because the value of activation energy

for this reaction is quite less compared to unanalyzed reaction. Due to

distraction in the bond-angles and bond lengths of stable configuration of

enzyme and substrate molecules after formation of enzyme-substrate

complex, the intermediate complex is destabilized.

It is not necessary that bonding between substrate and enzyme should

be strong to accelerate rate of the reaction. If there is no significant change

in bonding energy due to distortion strain, then substrate enzyme bonding

will be strong. If some free energy is used in distorting the substrate in the

transition state or in distorting the enzyme in the transition state, then

substrate-enzyme bonding will be weak.

8.4 EXAMPLES OF SOME TYPICAL ENZYME MECHANISMS

Although the mechanism of catalytic activity of enzymes is not

completely understood, much work has been done in this field using - (i)

specificity studies with the help of kinetic producers, (ii) analysis of active

400

centre of enzyme and (iii) comparative x-ray studies of complexes of some

enzyme with their sluggish substrates or competitive inhibitor's.

The enzyme whose mechanism of action is extensively studied

include chymotrypsin, ribonuclease, lysazyme and carboxyl peptides-A.

8.4.1 Chymotrypsin

Chymotrypsin is an important proteolytic enzyme i.e. it is a protein

hydrolyzing enzyme. Like, trypsin chmotrypsin is also secreted as an

inactive pro-enzyme, chymotrypsinogen, which is activated in the intestine

by trypsin, but not by enterokinase. Its optimum pH is between 8-9. It acts

in very much the same way as trypsin with a preferential attack on peptide

linkages involving the carboxyl group of typrosine and phenylalanine (Fig.

8.2). Moreover, like remain and pepsin, it has a powerful milk-clotting

action. The end-products of chymotrypsin hydrolysis are smaller

polypeptides and free amino acids. Trypsin hydrolyses residues of lysine and

orginine, while chymotrypsin hydrolyses residues only of aromatic amino

acids (Phe, Tyr and Try).

Fig. 8.2

401

Activity of chymotrypsin is supposed to be due to : (i) Position of His-

57 and ser-195 residues, near to one another on the active centre, and (ii)

Covalent acyl enzyme intermediate compound whose hydroxyl group of Ser-

195 takes part in the reactions.

The mechanism of hydrolysis of peptide bond by chymotrypsin is

explained as follows:

(a) Emidozole group of His-57 of chymotrypsin enzyme acts as a general

base.

(b) Hydrogen atom of -OH group of Ser-195 reside of Chymotrypsin and

nitrogen atom of emidezole ring of His-57 link through hydrogen

bond resulting in transfer of hydroxyl proton to nitrogen of His-57.

(c) In presence of a base, -OH group of Ser-195 under goes nucleophitic

attack on carboxylic carbon of aminoacyl group of the substrate and

forms acyl-chymotrypsin adduct.

In the second step of displacement, ser-195 acyl group acts as a donar

and water (alcohol, amino acid, amine) behave as an acceptor.

Out of the many mechanism, put forwarded for explaining activity of

Chymotrypsin on dipeptide, this base catalysed nucleophilic displacement

mechanism is an important one and cites an example of covalent catalysis.

8.4.2 Ribonuclease

The enzymes which degrade the nucleic acids are known as nucleases.

Some are specific for RNA and thus known as ribonucleases, and others for

DNA and thus known as deoxyribonucleaes, while still some others are

capable of attacking DNA as well as RNA.

402

ucleasedeoxyribonor

seRibonuclea

The pancreatic juice contains both the types of nucleases i.e.

ribonuclese (RNAase) and deoxyribonuclease (DNAase) or dornase. These

enzymes are endonucleases, i.e. they attack the molecule at sensitive

linkages in the interior of the chain. Ribonuclease hydrolyses RNA to

oligoribonucleotides as well as some pyrimidine ribonucleotide, while the

DNAase hydrolyses DNA to the corresponding oligonucleotide.

Nucleic acid Mononucleotides

(RNA or DNA)

Rbonuclease (i) use RNA as substrate,

(ii) follow endonucleolytic action,

(iii) attacks at site 1 (fig. 8.3) and thus leads to the

formation of 5-phosphoryl and 3-hydroxyl groups.

and (iv) works on single stranded and or double helical

condition of the substrate.

Fig. 8.3

On the whole ribonuclease degrades RNA to mononucleotides and

thus they may play a significant role in the destruction of nucleic acid of

viruses in the host cell.

403

8.4.3 Lysoenzyme

The enzyme Lysozyme (an antibiotic) found in human tears is used in

the therapy of eye infections. Large amounts of Lysozyme can be obtained

from hen egg white.

The crystalline form of the enzyme has been shown, by x-ray analysis,

to have the active centre in the form of long slit. Lysozyme forms enzyme-

substrate complex with some sluggish substrate or competitive inhibitor.

These sluggish or common substrate are linked with peptidoglycan, a

long chain proterin found in cell-wall of bacteria.

The enzyme-substrate complex gives following information as a result

of x-ray studies:

In the active centre -COOH group of glutamic acid and acid-52 group

of aspartic acid act as proton-donar and proton-acceptor respectively.

These concerted groups react using common acid-common base

catalytic mechanism,

The arrangement in the active centre has an another specialty that

glutamic acid-35 is oriented in such a manner that it is surrounded

from all sides by non-polar R groups, which enhance transfer of

proton.

404

8.4.4 Carboxypeptidase-A

The polypeptides obtained by the breakdown of proteins in the

intestine by ptyalin, trypsin and chymotrypsin enzymes are further attacked

by a mixture of two carboxypeptidases, A and B. These are secreted in their

corresponding proenzyme forms, which are activated by trypsin. They

apparently contain -SH groups, Mg and Zn are inhibited by iodoacetate,

cyanide, sulphide, cysteine, citrate, phosphate and oxalate. They are

exopeptidases and attack the peptide linkage of the terminal amino acid

possessing a free carboxyl group.

The two carboxypeptidases differ in the respect that the form A

attacks peptide linkages involving tyrosine, phenylalanine and tryptophan,

while the other form B attacks peptide linkages involving lysine and

orgainine.

Thus, carboxypeptidase A is an exopeptidase which hydrolyses C-

terminal peptide bond of polypeptide. The shape of its active centre is like a

pore or cavity, in which the C-terminal residue of polypeptide is fitted. In

405

this cavity are present Zn-atoms and R groups of Arg-145, Tyr-248 and Glu-

270.

X-ray analysis indicates that these groups are oriented in the active

centre, in such a way that C-terminal peptide bond is hydrolysed easily.

Check Your Progress - 1

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) According to Michaelis-Menton hypothesis.........................first

combines with....................to form............................which further

dissociates to give......................and....................back

(ii) Metals are supposed to serve two purposes in the enzyme subtract

interactions:

(a) .............................................

(b) .............................................

(iii)The biological responses of an enzyme is the result of its

interaction with receptor. Receptor may be considered as

a............on the.....................membrane.................with a..............

which facilitates its...................and.....................contact with

enzyme.

(b) (i) The bond cleavage rate is enhanced by the following moeds:

(a) .............................................

(b) .............................................

406

(c) .............................................

(ii) Chymotrypsin is an important.....................enzyme, i.e. it is

a.............................enzyme.

(iii)The important points in concern with the mechanism of

ribonuclease are:

(a) .............................................

(b) .............................................

(c) .............................................

(d) .............................................

8.5 Kinds of Reactions Catalysed by Enzymes

There are various types of biochemical reactions which are supposed

to be catalyse by enzymes. Some of more important reactions are given

below.

8.5.1 Nucleophilic Displacement on Phosphorus

Nucleophilic reagents or groups are electronrich species and have

tendency to react the atom of a compound or reagent. Which is electron

deficient. Nucleophilic displacement reactions involve displacement of a

nucleophile by another nucleophile:

Nu- + x - y x - Nu + y

-

Nuclophilic catalysis accelerates rate of a reaction and at the end of

the catalysis the nucleophilic catalyst is regenerated:

407

Uncatalysed Reaction: HXROHOHXR slow

2

Catalyzed Reaction: XRYYXR

eNucleophil

HXROHOHRY2

HXROHOHRX2

In covalent catalysis, catalyst takes part during the reaction

Nucleophiles are very effective and versatile catalysts. The enzyme

molecule may have three types of nucleophilic groups, which work as

catalyst:

(i) Hydroxyl group of serine: OHCH2

(ii) Sulphhydryl group of Cystine: SHCH2

(iii) Imadazole group of histadine: - CH2 C = CH

HN N:

CH

Some of the nucleophilic substitution reactions on phosphorous atom

are as follows:

(a)

In this reaction (a), covalent catalysis takes place, with the formation

of phospho-enzyme intermediate compound, due to nucleophilic attack on

phosphorous of the enzyme. The intermediate compound thus formed, loses

408

nucleophile -OR- group to give the product. Thus OR

- group is displaced by

X-.

(b)

Reaction (b) represents displacement of F- with ester group e.g. nerve

gas diisopropylfluoro phosphate (DFP) reacting with -OH group of an

enzyme gvies HF and O-phosphoryl ester.

This type of mechanism of enzyme catalysis is generally seen in the

reactions of transferase enzyme. Hexakinase is an important enzyme of this

type.

(c) For example, formation of fructos-1-phosphate from fructose in

presence of fructokinase:

(d) Similarly, transfer of phophoryl group from ATP to glucose is a

nucleophilic reaction, which takes place in presence of phosphato transferase

enzyme:

.D. Glucose .D-Glucose - -phosphate

409

8.5.2 Multiple Displacement Reactions and the Coupling of ATP

Cleavage to Endergonic Processes

In ATP, the phosphate group nearest to the ribose is termed as the

-phosphate group while the other phosphates are Labelled as and .

Adenosine diphosphate and adenosine triphosphate (ATP) (Fig. 8.4)

are involved in oxidative phosphorylation.

Fig. 8.4: Adenosine triphosphate (ATP)

In the cell, the conversion of ADP to ATP is used to store energy.

This energy can become readily available by the conversion of ATP to ADP.

This energy-producing system is a cyclic process in the cell.

OHATPmole/Kcal12POHADP243

mole/Kcal12POHATPOHADP432

Moreover, ATP is one of the most important compounds in the cell

since its two terminal phosphate groups are linked by the high-energy

phosphate bonds, i.e. it has two energy-rich bonds. It has a high potential for

410

group transfers. Depending on the nature of the bond of the ATP molecule

which reacts it can transfer following four different types of groups.

(a) Transfer of orthophosphate group with the release of ADP.

(b) Transfer of the pyrophosphate group with the release of AMP.

(c) Transfer of adenosyl monophosphate group with the release of

pyrophosphate (an activated compound).

(d) Transfer of adenosyl group with the release of orthophosphate as

well as pyrophosphate.

Fig. 8.5: Reactions of ATP.

Out of the above four reactions, reaction (a) is the most common. In

case the orthophosphate residue is transferred to water, hydrolysis results.

Enzymes catalyzing this reactions are known as adenosine triphosphatases

(ATPases). Reaction (b) (transferrence of a pyrophosphate group) occurs

rarely, one example is the conversion of ribose 5-phosphate to 5-

phosphoribose 1-pyroposphate (PRPP). The reaction (c) (transference of

411

adenosine monophosphate) is again quite common. The reaction (d) (transfer

of adenosyl residue) plays a part in the formation of the active methyl

groups.

It is important to note that ATP is not the only reactive triphosphate;

other purine, or pyramidine bases may take the place of adenine in the

molecule. The corresponding triphosphates replace ATP in several metabolic

reactions. In general, by analogy with AMP, ADP and ATP the nucleosides

guanosine, uridine, cytidine and inosine form GMP, GDP, GTP, UMP,

UDP, UTP, CMP, CDP, CTP, IMP, IDP and ITP.

The ATP in these processes becomes available to drive all those

processes which require energy; actually the number of examples where

ATP is utilized is as great as the number of types of physiological work

carried on by the cell; a summary of various processes involving endergonic

reactions is represented in fig. 8.6

Fig. 8.6: ATP as a common currency of bioenergetics.

Thus, ATP represents the energy currency of the cell.

412

Now let us discuss one example (synthesis of benzoyl glycine) to

show how ATP is used. The synthesis of benzoyl glycine (hippurice acid)

does not proceed sponstaneously as written below.

Benzoic acid + Glycine Hippuric acid + H2O

Acutally the synthesis occurs in the animal body by expenditure of

chemical work, i.e. it is endergonic.

Benzoic acid + ATP Benzoyladenlate + Pyrophosphate

(enzyme bond)

Benzoic acid + CO A Benzoyl- CO A + AMP

(enzyme bound)

Both the above reactions are reversible because in each case the bond

broken and the bond formed are energetically equivalent.

Now once the benzoyl-CoA is formed, it reacts with the amino group

of glycine to form hippuric acid.

Benzoyl-CoA + Glycine Benzoyl-glycine + CoA

This reactions is irreversible and takes place only in the right side

because the energy level of the thioester linkage in benzoyl-CaA is

considerably above that of a peptide or amide in benzoyl-glycine.

Similarly, in other examples where ATP is utilized, the same general

pattern is followed, i.e. a direct reaction between ATP and ths substrate

occurs, work is performed, and inorganic phosphate or pyrophosphate is

produced from the original high-energy phosphate bond. The same

mechanism is believed to occur in mechanical (as in muscle contraction),

413

osmotic (as in secretion, absorption and kidney functions), and electrical (as

in nervous impulses) work where ATP is utilized.

8.5.3 Addition and Elimination Reaction

Addition Reactions:

These reactions are given by unsaturated compounds in which the

reactant adds at the double bond resulting in a saturated compound:

A B

+ A – B ― ― ―

Unsaturated Saturated

Compound Compound

In biochemical activates, such type of reactions are common. For

example the reaction which takes place in citric acid cycle:

Fumerase

Fumerate (Trans) Melete (L)

This is an addition reaction which has specific dimensions, as the

trans isomer of the unsaturated dicarboxylic acid (fumerate) adds a water

molecule, at the double bond, to give L-isomer of -hydroxy dicarboxylic

acid (malete). The enzyme (fumerate) does not interact with as isomer of an

unsaturated acid and D-malete. The sterochemical studies using duteriated-

water indicated that the mechanism involves trans-addition of D+ and OD

- at

the double bond catalysed by the enzyme:

Fig. 8.7: Trans-addition of D2O

414

Similarly cis-aconitic acid which is an intermediate compound with

the enzyme (aconitase), add, a water molecule to give citric and isocitric

acids (on one side citric acid is formed and on the other side isocitric acid).

The isotopic studies, replacing H with D in the substrate indicate that H and

OH add trans at the double bond:

Citrate Cis-0Aconitic acid Isocitrate

The stereo specific trans addition of water may be represented as

follows:

Isocitric acid

Elimination Reactions:

415

Elimination reactions are just opposite to addition reactions. In

addition reactions, unsaturated compounds are concerted into saturated

compounds, while in elimination reactions saturated compounds are

converted into unsaturated compounds due to intra molecular elimination of

H2O, HCl or NH3 etc., e.g. in citric acid cycle succinic acid is oxidized in to

fumeric acid in presence of succinic dehydrogenase enzyme:

This is an example of 1, 2 or elimination reaction. In this reaction

succinate hydrogenase enzyme, trans-eliminating two hydrogens, gives

fumerate.

Similarly, enclose catalyzed glycosis also involves elimination

reaction:

2- Phospho- Phosphoric

Glycerate Pyruvate

In this reaction the unsaturated product is obtained due to elimination

of a water molecule from the substrate.

8.5.4 Enolic Intermediates Isomrisation Reactions: Isomerisation and

Rearrangement Reaction

Enolase

416

Isomerisation and rearrangement reactions play important part in

metabolism phenomenon. Isomerism is the reaction in which one isomer is

changed in to another. In biochemical reactions these transformations take

place in two ways:

(a) By change of position of double bond with the intermolecular change

in the positions of two hydrogen atoms, and

(b) Aldose-Ketose isomerism is an important example of intermolecular

shifting of hydrogen atoms e.g. inter conversion of dihydroxy acetone

phosphate into D-glyceraldehyde, 3-phosphate. This inter conversion takes

place in presence of triophosphate isomerase:

Similarly, the reversible reaction which takes place in presence of

glucose phosphate isomerase :

Dihydroxy d. Glyceraldehyde-3-phosphate

acetone phosphate

Furctose - 6 - phosphate Glucose - 6 - Phosphate

In this reaction enediol is the intermediate compound :

417

This reaction involves, conversion of C1-carbonyl group of glucose

into C2-carbonyl group of fructose. In the next step, OH group attached to C1

of fructose is easily phosphorylised to give 1, 6 diphosphate.

(b) Intermolecular rearrangement of functional groups is comparatively

less frequent in metabolic processes. However, in metabolism of

glucose, conversion of -D - glucose-6-phosphate into -D - glucose-

1-phosphate in presence of phosphoglucomerase is an important

example of the phenomenon. In this isomerisation reaction phosphate

group present on carbon-6 is shifted to carbon-1 :

Pentose phosphate also shows different types of isomerisation:

-D.Glucose- -D-Glucose

b-phosphate 1-phosphate

Phsophoglucomutase

epimerase

Ribulose-

5- Phosphate

Zybulose 5-Phosphate

418

The epimerase enzyme making change in the arrangement at carbon-3

gives the epimer, hence the process is known as epimerization (a special

type of isomerism).

Ribulose-05 Enediol Ribose-5

Phosphate Phosphate

In the reaction a Ketose is converted into an aldose.

8.5.5 Condensation Reactions

Condensation is the process of combination of two or more molecules,

with elimination of water or ammonia molecule, to give compound of higher

molecular weight. The simplest example of the process is condensation of

two molecules of acetaldehyde, in presence of dilute alkali to give aldol

(Aldol condensation):

Aldehyde Aldol

During metabolic process of biomolecules, generally aldol and

retroaldol reactions are seen frequently. For example, the citrate synthase

catalysed reaction in first step of cirtic acid cycle, the first intermediate

compound, citric acid cycle, is formed due to condensation of oxaloacetic

acid and acetyl co enzyme A :

419

Succinic acid

In this reaction aldol condensation takes place when hydrogen atom of

methyl group in acetyl-co-enzyme A combines with carbonyl oxygen of

oxaloacoetate. Aldol is the intermediate compound.

Similarly, when D glyceradehyde 3-phosphate and dihydroxy acetone

phosphate condense to give -D-frucotse, 1, 2 diphosphate, in the front side

aldol condensation and in the back side retro aldol condensation takes place.

The enzyme in this reaction is aldolase:

8.5.6 Enzyme Catalyzed Carboxylation and Decarboxylation

Carboxylation and decarboxylation are reversible reactions. In

metabolic processes both these reactions are seen frequently, e.g. formation

of oxalo acetic acid from pyruvate in presence of an enzyme involves

carboxylation:

Mn2+

pyruvate + CO2 + ATP + H2O oxaloacctate + ADP + Pi

420

Thus in an important reaction which is catalysed by pyruvate acid

and hydroxy acid take part in different metabolic processes, hence

decarboxylation reaction is quite common in these processes.

For example, formation of acetaldehyde from pyruvate in the presence

of pyruvate decarboxylate enzyme present in yeast cells:

CH3

C O CH3CHO + CO2

Acetaldehyde

COOH

Similarly, in citric acid cycle, the first step is formation of

oxalosuccinic acid, as an intermediate compound, due to dehydrogenation of

isocitrate, which being unstable readily decarboxylate to give the product,

-Ketoglutarate. This is an example of Oxidative decarboxylation which

takes place in two steps:

Check Your Progress - 2

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) Nueleophiles are very....................and.......................catalysts and

are supposed to have following three types of nuclephilic groups:

(i) ...............................

(ii) ...............................

(iii) ...............................

421

(ii) Depending on the nature of the bond of the ATP molecule, it can

transfer following four different types of groups:

(a) ...............................

(b) ...............................

(c) ...............................

(d) ...............................

(iii)Aldose-Ketose isomerism is an important example of.............

shifting of...............atoms e.g. interconversion of..............into D-

glyceraldehyde-3-phosphate.

(iv)Formation of oxaloactive acid from...................in presence

of..............enzyme present in...................is an important.............

reaction.

8.6 LET US SUM UP

After going through this unit you must have achieved the objective

stated at the start of the unit. Let us recall what we have discussed so for:

Enzymes are biocatalyst, synthesized by the living cells. Enzymes

have immense catalytic power and accelerate reactions at least million

times, by reducing the energy of activation.

All the enzymes are protein in nature with large molecular weight.

While a few are conjugated protein. In such enzymes the non-protein

part is called prosthetic group or coenzyme and the protein part is

called as apoenzyme.

422

According to Michaclis-Menten hypothesis, enzyme molecule (E) first

combines with a substrate molecule (S) to form an enzyme-substrate

complex (ES) which further dissociates to form product (P) and

enzyme (E) back.

Enzyme activity depends upon the presence of various metals. Three

probable mechanisms have been proposed to explain the mode of

action of enzyme:

(a) Metal ion, M, forms complex with enzyme-protein (E) to give

active enzyme (EM):

E + M EM

The active enzyme reacts with the substrate to give an

intermediate, enzyme-metal-substrate complex (EMS). Which

subsequently decomposes into product, P, and the active enzyme:

EMS EM + P

(b) The metal ion forms complex with substrate (MS) first which

reacts with enzyme to give enzyme-metal-substrate complex. Which

subsequently decompose into product, the enzyme and the metal:

M + S MS

E + MS EMS

EMS P + E + M

(c) The enzyme (E) reacts with the substrate (S) to give ES

complex. Which combines with metal to give enzyme substrate metal

complex (ESM). This finally decomposes to furnish the product:

M + S ES

423

ES + M ESM

ESM P + S + M

Metals are supposed to serve two purposes: (i) provide proper stereo

chemical orientation and (ii) bring reacting molecule (the enzyme and

substrate closer so that the reaction may occur.

There is a group of enzymes which are involved in the natural process

of bond-formation and bond-cleavage. During these reactions many

modes are involved which enhance rate of bond cleavage viz:

(a) Acid-base catalysis.

(b) Covalent Catalysis, and

(c) Strain and molecular distraction

The specific acid-base catalysis reactions are those reactions, the

rates, of which are changed with the change in concentration of H+ or

H3+O ions in the solution; while they remain unaffected in the

presence of other acids and bases.

The formation of covalently bonded intermediate complex results,

due to nucleophilic attack of enzyme on substrate. Acylation,

phosphorylation or glycolysation of nucleophile are examples of

covalent catalysis.

Mechanism of catalytic activity of enzyme is studied (i) using kinetic

procures (ii) analysis of active centers of enzyme, and (iii)

comparative x-ray studies of complexes of some enzymes with their

sluggish substrates or competitive inhibiters.

424

The enzymes whose mechanism of action is extensively

studied include chymotrypsin, ribonuclease, lysozyme and

carboxypeptidase-A.

Chymotrypisn hydrolyses aromatic amino acid residues. Ribonuclease

degrades RNA to mono nucleotides. While lysozyme found in human

tears is used in the therapy of eye infections. The carboxyl peptidase

attacks peptide linkages involving tyrosine, phenylalanine and

tryptophan. It is an exopeptidase which hydrolyses.

Some of the more important biochemical reactions, catalyzed by

enzymes include (i) Nucleophilic displacement reactions on

phospharous atom,

(ii) ATP cleavage to endergonic process,

(iii) Addition and elimination reactions.

(iv) Condensation reactions,

(v) Isomerisation and rearrangement reactions, and

(vi) Carboxylation and decarboxylation reactions.

8.7 CHECK YOUR PROGRESS: THE KEY

1 (a) (i) enzyme molecule (E)

substrate molecule (S)

Enzyme-substrate complex (ES)

product

enzyme

(ii) (a) bring reacting molecules closer and

(b) provide proper stereochemical orientation

425

(iii) locus

protoplasmic

structure

preferential

selective

(b) (i) (a) Acid-base catalysis

(b) covalent catalysis, and

(c) strain and molecular distortion

(ii) proteolytic

protein hydrolyzing

(iii) (a) use RNA as substrate

(b) follow endonucleolytic action

(c) attack at site1 to form 5-phosphoryl and 3-

hydroxyl groups.

(d) work on single strand and/or double helical

condition of the substrate.

2. (a) (i) (a) Hydroxyl group of serin, -CH2OH

(b) sulph-hydryl group of cystine, -CH2SH

(c) Imadozole group of histadine, ―CH2 – C = CH

HN N

CH

(ii) (a) orthophosphate group with release of ADP.

(b) the pyrophosphate group with release of AMP.

(c) Adenosyl monophosphate group with release of

pyrophosphate.

426

(d) Adenosyl group with release of orthophosphate

and pyrophosphate.

(iii) intramolecular hydrogen

dihydroxy acetone phosphate

(iv) pyruvate

pyruvate carboxylate

mitochondria

carboxylation

UNIT-9 COENZYMES

Structures

9.1 Introduction

9.2 Objectives

9.3 Co-Enzymes: Co-Factors

9.4 Some Important Co-Enzymes

9.4.1 Co-Enzymes - A (Pantobenic acid)

9.4.2 Co-Enzymes - B1 (Thiamine Pyrophosphate)

9.4.3 Co-Enzymes - B6 (Pyridoxal Phosphate)

9.4.4 Co-Enzymes - Niacin (NAD and NADP)

9.4.5 Co-Enzymes - Riboflavin (FAD and FMN)

9.4.6 Co-Enzymes - Lipoic Acid

9.4.7 Co-Enzymes - B12 (Cyanocobalamine)

9.5 Enzyme Models

9.5.1 Host Guest Chemistry

9.9.2 Chirality and Catalysis

9.5.3 Molecular Asymmetry and Prochirality

9.5.4 Biomemetic Chemistry

(a) Crown-Ethers and Cryptates

427

(b) Cyclodextrins and Cyclodextrin based enzyme model (c)

Calixarenes

(d) Ionosphere

(e) Micelle

9.6 Synthetic Enzyme or Syn-Enzyme

9.6 Let Us Sum Up

9.7 Check Your Progress: The Key

428

9.1 INTRODUCTION

In general, with exception of ribozymes which are a few RNA

molecules with enzymatic activity, all the enzymes are proteins in nature

with large molecular weight. Some enzymes are simple proteins while some

are conjugated proteins. In such enzymes the non-protein part is called

prosthetic group or Co-enzyme and the protein part is called apo-enzyme.

The complete structure of Apo-enzyme and prosthetic group is called

holoenzyme:

Holenzyme = Apo-enzyme + Co-enzyme

(Protein part) (Prosthetic group)

The non-protein part or prosthetic group of enzyme i.e. conjugated

protein is essential for reactivity of protein. In other words, when enzyme

and coenzymes are together then only their catalytic activity become

evident. Thus the main characteristics of Co-enzyme are:

(i) They are non-proteins.

(ii) They are not destroyed by heat.

(iii) They can easily be separated from protein part of the enzyme

by dialysis.

(iv) These are organic compound.

(v) Their molecular weights are quite low.

Co-enzymes are also known as co-factors and take part in different

reactions, such as redox, group transfer isomerisation etc and form covalent

bonds. On the basis of their functions and the vitamins obtained from them,

cofactors are classified as follows :

429

I. Co-enzymes which give nicotinamide and function as hydrogen

carrier:

(i) Nicotinamide Adenine di-nucleotide (NAD).

(ii) Nicotinamide Adenine dinucleotide phosphate (NADP).

II. Co-enzymes which give riboflavin and function as hydrogen

carrier:

(i) Flavin mononucleotide (FMN).

(ii) Flavin adenine nucleotide (FAD).

(iii) Lipoic acid (Lip(S)2)

(iv) Cytochrome (Cyt)

III. Co-enzymes which give thiamine:

(i) Thiamine Pyrophosphate (TPP).

(ii) Uridive diphosphate (UDP).

(iii) Uridine triphosphate (UTP).

IV. Co-enzymes which give pantathenic acid:

(i) Co-enzyme - A (COASH).

(ii) Cytodin diphosphate (CDP).

In these Co-enzyme some of Co-enzyme are involved in trans

phosphorylation (ATP, CDP), some acyl group transfer (COASH) and some

in monosaccharide formation (UDP, UTP).

9.2 OBJECTIVES

430

The main aim of the unit is to discuss importance of various co-

enzymes and enzyme models. After going through this unit you would be

able to:

explain cofactors or co-enzymes,

describe importance of various co-enzymes and the vitamins obtained

from them,

discuss various enzyme models such as Host-Guest model, and

understand what are Syn-enzymes?

9.4 SOME IMPORTANT COENZYMES

As has been pointed out the dialyzable part of a conjugated protein is

termed as coenzyme, the protein part of a conjugated protein as Apo-enzyme

and the intact molecule or conjugated protein as holoenzyme.

Thus coenzyme may be defined as a substance necessary for the

activity of the enzyme. However, biochemists now use the term co-factors,

in this general way and specify coenzyme only for those cofactors which are

organic molecules and participate in the catalytic process, for example in

metalloprotein enzymes, the metallic ion is not the coenzyme, but only

organic prosthetic group constitutes the coenzymes. We may say every

coenzyme is a cofactor but every cofactor is not a coenzyme. Most of the

coenzyme are nucleotides and are composed of vitamins.

9.4.1 Co-Enzymes - A (Pantothenic acid)

Pantothenic acid consists of -alanine in peptide linkage with a di-

hydroxy di-methyl butyric acid ('Pantoic' acid).

-alanine + Pantoic acid Pantothenic acid

431

Occurrence and Food Sources:

It is widely distributed in plants, animal tissues and food materials:

Excellent food sources: (100 to 200 g/gm of dry materials)

including kidney, liver, egg-yolk and yeasts, cereals and legumes.

Fair sources: (35 to 100 g/gm) including skimmed milk, chicken,

certain fishes, sweet potatoes, molasses.

Most vegetables and fruits are rather poor source.

Richest known source of pantothenic acid is Royal Jelly (also rich in

Biotin and Pyridoxine).

Note: A 57% loss of pantothenic acid in wheat may occur during the

manufacture of patent flour and about 35% is lost during the cooking of

meat.

Its biological active from is coenzyme A: In tissues, this vitamin is present

almost entirely in the form of the coenzyme (co-enzyme A is also known as

Co-acetylase) and largely bound to proteins (apoenzyme). It may be released

from this combination by certain proteolysis enzymes, certain phosphates,

and a liver enzyme system.

Biosynthesis and Metabolism:

I. Biosynthesis of Pantothenic Acid:

(a) In many microorganisms, including yeast pantothenic acid is

synthesized by direct coupling of -alanine and pantoic acid -Alanine

is formed from decarboxylation of Aspartic acid and pantoic acid from

-keto isovalerate.

432

(b) Human tissues cannot synthesize pantothenic acid hence it has to be

obtained from diet. In addition to dietary source, synthesis by intestinal

bacteria supply fair amount of pantothenic acid.

II. Synthesis of Coenzyme-A: Complete synthesis of coenzyme A was

described by Khorana in 1959. Human tissues as well as plants and

bacteria can synthesize CoA-SH. Synthesis of coenzyme A is shown

schematically in box ahead in next page.

The free acid is soluble in water and is hydrolyzed by acids/or alkalis.

It is thermolabile and destroyed by heat.

It is yellow coloured syrupy and oily liquid

Structure of Coenzyme A: Structure of coenzyme A has been

delineated and can be represented schematically as follows (Fig. 9.1).

Fig. 9.1: Structure of Cofactor-A

Pantotheruc acid is joined in one hand to adenosine-3'-P by a

pyrophosphate bridge, and on the other hand.

433

Joined by peptide to -mercaptoethanole amine, which is obtained

from ammo acid cysteine. The terminal-SH group (thiol group) or -

mercaptoethanole amine is the reactive site of the coenzyme molecule

("Active site" or group).

Note: For convenience co-enzyme A is represented as CoA-SH. The

naturally occurring forms of the coenzyme probably include:

The reduced -SH form,

The oxidized -S-S-forms, and

Combinations of the -SH group with various metabolites, e.g.. acetate,

and succinate to form acetyl CoA and succinyl CoA respectively.

Functions: As discussed earlier pathothenic acid is a component of

coenzyme A. Coenzyme A (COA-SH) in turn has a key role in metabolic

processes, where its main function is the transfer of acyl groups (e.g. acetyl

butyryl, succinyl) in both catabolic acid biosynthetic reactions. For example,

acetylocenzyme A is involved in the conversion of oxaloacetic acid to citric

acid in the Kreb's cycle.

Actually coezyme A plays an essential role in the metabolism of fats

and carbohydrates.

434

Coenzyme A and hence pantothenic acid is also involved in the

formation of cholesterol and adrenocortical hormones from active acetate

(acetyl-CoA).

The symptoms of pantothenic acid deficiency in man are unknown

partly due to its widespread distribution in foodstuffs and partly due to its

synthesis (although to a limited degree) by intestinal bacteria. However,

this vitamin is necessary for chicks and rats. Deficiency of the vitamin in

chicks causes a specific dermatitis, retardation and toughness of feathers. It

also helps for hatchability and reproduction in domestic fowl. The deficiency

of the vitamin in rats causes retardation of growth, depigmentation and

spectacled condition of the age.

9.4.2 Co-Enzymes - B1 (Thiamine Pyrophosphate)

It is one of the longest known vitamins. Chemically, it consists of a

pyrimidine and a thiazole ring system joined by a methylene bridge. It is

generally prepared as a chloride hydrochloride.

Chemically it is 2, 5-dimethyl, 4-methyl-6-amino pyrimidine 5-OH

ethyl thiazole, Thiamine pyrophosphate (thiamine diphosphate). Its structure

was determined by Willioms and associated (Fig. 9.2)

Biological "active" from is Thiamine Pyrophosphate (TPP): Acts as a

coenzyme in several metabolic reactions:

Acts as coenzymes to the enzymes pyruvate dehydrogenate complex

(PDH) which converts pyrucvic acid to acetyl CoA (oxidative

decarboxylation)

435

Similarly acts as a coenzyme to -oxoglutarate dehydrogenase

complex and converts -oxoglutarate to succinyl CoA (oxidative

decarboxylation).

TPP also acts as a coenzyme with the enzymeTransketolase in

transketolation reaction in HMP pathway of glucose metabolism:

B1 is also required in amino acid Tryptophan metabolism for the

activity of the enzyme Tryptophan Pyrrolase.

Also acts as a coenzyme for mitochondrial branched-chain -keto

acid decarboxylase which catalyzes oxidative decarboxylation of

branched-chain -keto acids formed in the catabolism of valine,

Leucine and iso-leucine. TPP binds with and decarboxylates these

branched chain -keto acids and transfers the resulting activated -

CHO groups to -lipoic acid.

TPP acts as the coenzyme (Co-carboxylase) of pyruvate carboxylase

in yeasts for the non-oxidative decarboxylation of pyruvate to

acetaldehyde.

Fig. 9.2: Structure of Thiamine Pyrophosphate

Sources: Thiamine is widely distributed throughout the plant kingdom, in

particularly high concentration in the seeds. In cereal grains, it is

concentrated in the outer germ and bran layers (e.g., rice polishing). Which

436

are often discarded during milling processes (e.g. of wheat flour and rice).

Thus ordinary white flour contains little thiamine, while the whole meal

flour is a good source of the vitamin. Pulses and nuts are among the richest

natural sources of the vitamin. Thus the following constitute good dietary

sources of the vitamin: peas, beans, whole cereal grains, bran, nuts, prunes,

gooseberries, killed yeast.

Thiamine is also present in most animal tissues and the lean parts of

meat, especially pork, are important dietary sources. Beef mutton, liver, and

eggs also supply considerable amounts Milk, although contains

comparatively low amounts, it being taken in large amounts constitutes an

important dietary source.

The body cannot store large amounts of thiamine. The total amount of

the vitamin in the body is about 25 mg. the heart has the highest

concentration followed by brain, liver, kidney, skeletal muscle and blood.

It is soluble in water (1 gm./l ml.) and 95 of ethanol (1gm./100ml.) but

not in fat solvents. It is resistant to heat, (boiling or autoclaving) in solutions

below pH3.5

, but becomes inactive above pH 5-5 owing to hydrolysis. The

thiamine content of the vegetables may be preserved by freezing and by

storing below 0oC. It is not oxidized by atmospheric oxygen under ordinary

conditions but mild oxidizing agents oxidize it to the inactive pigment

known as trichrome. Owing to its basic. characters, it forms, a number of

salts and esters; the most important ester being the thiamine pyrophosphate

(TPP or co-carboxylase). TPP is formed by the phosphorylation of the

vitamin under the influence of ATP and magnesium ions.

437

Free thiamine (but not the TPP) is absorbed readily from the small

intestine. Most of the dietary vegetable thiamine is in the free state, it is

phosphoryiated to TPP mainly in the liver. Under normal dietary amounts

(1-2 mg. daily) about 10 percent of the daily dietary allowance is excreted in

the urine. It is secreted in the milk as a thiamine-protein complex and, in

certain species (e.g., goat), as mono and di-phosphothiamine.

Functions: The physiological actions of thiamine are due to its

pyrophosphate known as thiamine pyrophosphate (TPP), or

diphosphothiamine (DPT) or co-carboxylase. TPP, together with -lipoic

acid, is coenzyme of the enzymes carboxylases needed for the

decarboxylation of a-keto acids such as pyruvic and -ketoglutaric acids.

Thus when thiamine is lacking, pyruvic acid accumulates in the fluids and

tissues of the body. TPP is also involved in certain transketolase reactions,

especially those of the pentose pathway of photosynthesis. Deficiency of this

vitamin leads to disruption of carbohydrate metabolism.

Thiamine deficiency affects mainly the peripheral nervous system, the

gast ointestinal tract, and the cardiovascular system. Prolonged deficiency of

the vitamin in man results in the condition know as beriberi. Beriberi, still

common in many parts of the world, is characterized by three groups of

systems: (a) polyneuritis, that is to say muscle weakness and atrophy,

incoordination of movements, and disturbances of sensation;

(b) enlargement of the heart and cardiac failure ; (c) dropsy or edema.

9.4.3 Co-Enzymes - B6 (Pyridoxal Phosphate)

Actually the word vitamin B6 refers to a group of three compounds

namely, pyridoxine, pyridoxal or adermin; pyridoxal; and pyridoxamine

438

which arc interconvertible in the form of their phosphates, All the Three

compounds exhibit vitamin activity, which, however, actually resides

apparently in phosphorylated derivatives. But as pyridoxine is the first

member of this group it is alone also known as vitamin B6. As the vitamin is

antidermatitic factor for rats it is also known as adermin.

Biological 'active' forms of the vitamin are:

Pyridoxal-PO4 and,

Pyridoxamine-PO4

In phosphate derivatives, the hydroxymethyl group of C5 is

phosphorylated:

Pyridoxyl Phosphate

Occurrence: It is found both in the Plant as well as in animal kingdom.

Mostly it is found in the combined form with protein (apoenzyme) and

starch but sometimes in the free form also, e.g. yeast and fish muscle. .

Most abundant sources for the vitamin are yeast and rice polishing

followed by seeds and cereals viz. wheat, maize, etc. Fish liver, kidney,

milk, fresh vegetable, egg and meat also contain small amounts of vitamin

B6.

439

Pyridoxal and pyridoxamine are secreted in milk in very small

amounts. Although both of these compounds are also excreted in the urine in

small amounts (0.5-0.7 mg. daily), the major urinary metabolite is the

biologically inactive 4-pyridoxic acid (about 3 mg. daily). In case either of

the three compound having vitamin activity are administered in large

amount 30-70% of the administered dose may be excreted unchanged in

urine.

Functions: All the three members of the group are active in biological

activity because these are interconvertrble but the most active is pyridoxal

phosphate obtained by phosphorylation (ATP) of pyridoxal. These are

involved in a number of important metabolic reactions of the -amino

acids, e.g., transamination, racemization, decarboxylation and elimination

reactions.

Pyridoxal P acts as a coenzyme, it is principally involved with

metabolism of amino acids.

Co-transaminase: Acts as a coenzyme for the enzyme transaminases

(aminotransferease) in transamination reaction.

440

Co-decarboxylase: Acts as coenzyme for the enzyme decarboxylases

in decarboxylaion reaction. Amino acids are decarboxylated to form

corresponding amines.

Examples:

Tyrosine Tyramine + CO2

Histidine Histamine + CO2

Glutamic acid G A B A + CO2

Acts as coenzyme for dreaminases (dehydrases) catalyes non-

oxidative deamination of OH amino-acids viz. serine, threonine etc.

Coenzyme for kynureninases: In tryptophan metabolism, pyridoxal-P

acts as coenzyme for the enzyme kynurcninase which converts 3-OH

kynurenine to 3-OH and anthranilic acid which ultimately forms

nicotinic acid. Thus in B6-deficiency niacin synthesis from tryptophan

does not take place. In B6 deficiency, kynurenine and 3-OH

kynurenine levels increases and they are converted to 'xanthurenic'

acid in extrahepatic tissues, which is excreted in urine. Xanthurenic

and 'index is a reliable criterion for B6 deficiency. Examination of

urine for xanthurenic acid after the feeding of a test does of

tryptophan has been used to diagnose B6 deficiency.

Transulfuration: It takes part in transulfuration reactions involving

transfer of -SH groups e.g.

Homocysteine + Serine homoserine + cysteine

As coenzyme for desulfhydrases: Catalyzes non-oxidative

deamination of cysteine in which H2S is liberated.

441

In inter conversion of Glycine and sering by serine hydroxy methyl

transferase in this both FH4 and B6 are required as coenzymes.

Pyridoxal-P is required as a co-enzyme in the biosynthesis of

arachidoinc acid form 'linoleic acid."

Synthesis of Sphingomyelin: Pyridoxal-P is required as a coenzyme

for activation of serine which is required for synthesis of

sphingomyelin.

Required as a coenzyme for amino acid racemases :

D-Glutamic acid L-Glutamic acid

D-Alanine L-Alanine

Intramitochondrial FA synthesis: Required as a coenzyme with

condensing enzyme for chain elongation of F.A. in intramitochondrial

F.a. synthesis.

Required for "active transport" of amino acid through cell membrane

and intestinal absorption of amino acids.

Muscle phosphorylase: As a constituent of muscle phosphorylase : 4

molecules of pyridoxal-(P) per molecule of enzyme (tetramer).

Transport of K+; Vitamin B6 has been reported to pro-mole transport

of K+ across the membrane from exterior to interior.

As coenzyme for aminoacetone synthetase which is required for

formation of amino acetone forms acetyl CoA and glycine.

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Synthesis of CoA-SH (Coenzyme A); Vitamin B6 is involved in

synthesis of coenzyme A from pantothenic acid. In B6 deficiency

coenzyme A level in liver is reduced.

9.4.4 Co-Enzymes - Niacin (NAD and NADP)

Nicotinic acid (niacin) is chemically Pyridine-3-carboxylic acid.

In tissues: occurs principally as the 'amide' (nicotinamide,

niacinamide). In this form it enters into physiological active combination.

In tissues, nicolinamide is present largely as a "inucleotide" the

pyridine 'N' being linked to a D-ribose residue. Two such nucleotide forms

are known :

(1) Nicotinamide adenine dinucleotide (NAS+): Other names is are:

DPN+, coenzyme-1, cozymase or codehydrogenase. The compounds

contains :

One molecule of nicotinamide.

Two molecules of D-ribose.

Two molecules of phosphoric acid, and

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One molecule of adenine. Structure may be shown schematically as

follows;

NAD

(2) Nicotinamide adenine dinucleotide phosphate (NDPF): Other

names are TPN+, co-enzyme II.

This compound differs from NAD+ in that it contains an additional

molecules of phosphoric acid attached to 2-position of D-ribose attached to

N-9 of Adenine. The reduced from of either coenzyme is designated by the

prefix "dihydro". e.g. reduced NAD is called "dihydro-nicotinamide adenine

dinucleotide (NADH).

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NADP

Nicotinamide and its derivatives (NAD and NADP) are widely

distributed in plant and animal tissues, the latter contain only the combined

nicotinamide (NAD and NADP). Like other vitamins of B group,

nicotinamide is present in highest concentration in the germ and pericarp

(bran), which are often discarded in the milling process (e.g. wheat and rice).

Yeast, and beer are good sources of the vitamin. Among the animal tissues

liver, kidney and muscles are the best sources of the vitamin. Thus the

important food sources of the vitamin are bran, yeast, liver, kidney, meats,

fish, barley, maize, nuts, peas, beans, whole-wheat, artificially enriched

white bread, certain green vegetable, coffee and tea. Fruits, milk and egg are

usually poor sources. Since nicotinic acid is very stable, loses in the

preparation and cooking of food are small; however significant amount may

be lost into the cooking water during extraction.

Nicotinic acid and its amide are absorbed from the intestine. Small

amounts of nicotinamide are secreted in milk and perspiration. Although

445

nicotine acid and nicotinamide are excreted in the urine in the free (0.25-

1.25 mg; and 0.5-4 mg. daily respectively) viz. N-methyinlcotinamide and

the 6-pyridone of N-methylnicotinamide, and N-Methyl-nicotinic acid and

the glycine conjugates of these methyl derivatives. The process of

methylation and oxidation occur in the liver.

Biosynthesis:

Amino acid Tryptophan is a precursor of nicotinic acid in many plants

and animal species including human begins. 60mg of tryptophan can

give rise 1 mg of Niacin. Pyridoxal-P is required as a coenzyme in

this synthesis.

Can be synthesized also by intestinal bacteria, Bacteria in addition to

synthesis from tryptophan, can also synthesize from other amino acids, e.g.

glutamic acid, proline, ornithine and glycine.

In human beings:

In addition to dietary source.

It is synthesized in tissues from amino acid tryptophan, and

to a limited extent supplemented by bacterial synthesis in intestine.

Synthesis of NAD+ and formation of NADP: Synthesis of NAD

+/and

NADP+ is shown above diagrammatically in Fig. 9.3

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Fig. 9.3

Biological functions: Nicotinamide is a part of the structure of

coenzyme 1 (DPN or NAD+) and coenzyme II (TPN or NADP+). These are

the hydrogen transferring coenzymes which is due to the presence of

nicotinamide moiety. They accept hydrogen from the substrate (which is

oxidized) and then transfer step by step to other compounds such as

flavoproteins, cytochrome b, cytochrome c, cytochrome a and cytochrome

oxidase (electron transport chain). The last compound gives us hydrogen to

oxygen, forming water. The overall result is the oxidation of the substrate. In

this way NAD and NADP function as coenzymes for a large number of

dehydrogenases involved in the metabolism of carbohydrates, Iipids, amino

acids and -keto acids. Transference of hydrogen is reversible and stereo

specific.

Some 40 or more biochemical reactions, including the synthesis of

high-energy bonds, glycolysis, pentose metabolism, lipid metabolism, and

pyruvate metabolism, require the coenzyme I or II.

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Some 40 or more biochemical reactions, including the synthesis of

high-energy bonds, glycolysis, pentose metabolism, lipid metabolism, and

pyruvate metabolism, require the coenzyme I or II.

Deficiency of nicotinic acid in human leads to the condition called

pellagara (pelle=skin; agra=rough). Pellagra is a special type of dermatitis

followed by malfunction of digestive, nervous, and brain tissue, and

ultimately death. This disease affects many millions of people every year,

especially in maiz eating areas where extreme poverty allows only a very

inadequate diet. The symptoms of the disease at an early stage are weakness,

lassitude, and indigestion. However, the chief symptoms of the acute

pellagra have been referred to as three "D's." i.e., dermatitis, diarrhea, and

dementia.

The skin of those parts of the body, which are normally subjected to

the action of light and heat, (e.g. face, neck, dorsal surfaces of the wrists and

forearm, the elbows, knees, etc.) becomes reddened, later brown, roughened,

and scaly.

Gastrointestinal manifestations include anorexia, nausea, vomiting,

abdominal pain, with alternating constipation and diarrhea. The mouth

becomes exterme red and sore and the tongue swollens and becomes red.

General effects of the deficiency of the vitamin include inadequate

growth of children, loss of weight and strength, anemia, and dehydration and

its consequences.

The two coenzyme are interconvertible. The important enzymes to

which NAD+ and NADP

+ act as coenzyme are given below in the table 9.1

Table 9.1

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Alcohol dehydrogenase (Ethanol

Acetaldehyde)

Glucose-6-P-dehydrogenase

(G-6-PD)

Lactate dehydrogenase (LDH)

(P.A.L.A.)

(G-6-P6-

Phosphogluconate)

Malate dehydrogenase

(MalateO.A.A.)

Gluathione reductase

Glycereaidehyde-3-P-

dehydrogenase (Gly-3-P1, 3-di-

phosphoglycerate)

-Glycero-P- dehydrogenase

Pyruvate dehydrogenase complex

(PDH)

(P.A. Acetyl CoA)

-Ketoglutarate dehydrogenase

complex

( -ketoglutaratesuccinyl CoA)

Either NAD+ or NADP+

Glutamate dehydrogenase

(Glutamate -

ketoglutarate+NH3)

Isocitrate-dehydrogensase (I

C D)

(IsocitrateOxalosuccinate)

9.4.5 Co-Enzymes - Riboflavin (FAD and FMN)

Riboflavin is an orange-yellow compound containing.

(i) a ribose alcohol: D-Ribitol and

(ii) a heterocyclic parent ring structure 'Isoalloxazine" ('Flavin' nucleus). 1-

Carbon of ribityl group is attached at the 9 position of iso-alloxazine

nucleus. Ribityl is an alcohol derived from pentose sugar D-ribose.

It is stable to heat in neutral acid solution but not in alkaline solutions.

Aqueous solutions are unstable to visible and UV light. The reactions are

irreversible.

Riboflavin undergoes reversible reduction really in presence of a

catalyst to a colourless substance "Leucoriboflavin."

449

The biological "active" forms in which riboflavin serves as the

prosthetic group (as coenzyme) of a number of enzymes are the

phosphorylated deivatives.

Two main derivatives are:

(a) FMN (Flavin mononucleotide): In this the phosphoric acid is affected

to ribityl alcoholic group in position 5.

Flavin - Ribityl - PO4

(b) FAD (Flavin adenine nucleotide): It may be linked to an ademine

nucleotide through a pyrophosphate linkage to form FAD.

Flavin - Ribityl - P - P - ribose - Adenine

Thus, FMN and FAD are two coenzyme of this vitamin.

FMN

450

FAD

The acidic properties given by phosphoric acid group influence their

capacity for combining with proteins apoenzyme-forming "flavo-proteins"

(Holoenzyme) . Thus,

Fp (holoenzyme) = FMN /FAD + Protein

(co-enzyme) (Apo-enzyme)

Fp may also unite with metals like Fe and Mo thus forming "Metallo-

flavoproteins".

In the living cell, riboflavin is converted into riboflavin phosphate

(flavin mononucleotide, FMN), and flavin adenine dinucleotide (FAD), both

of which combine with proteins to form the flavoproteins which in turn act

as important hydrogen carries in biologial oxidation systems.

Sources: Riboflavin, being an essential component of many biological

oxidation-reduction systems, is present nearly in all plant and animal cells.

Comparatively high concentrations are found in yeasts and fermenting

bacteria. Among the food-stuffs the most important source is milk.

Appreciable amounts of the vitamin are found in liver (2-3 mg. per 100

gm.), kidney, heart, crab meat, whole grain, dry beans and peas, nuts, milk,

eggyolk, meat, and green leafy vegetables. In yeast and most plant and

451

animal tissues, it is present mainly in the combined from (e.g., as FMN and

FAD) while in milk it is found mainly in the free state.

Ordinary cooking processes cause little destruction of riboflavin.

Considerable loss of the vitamin may occur in milk exposed to bright

sunlight.

Riboflavin is absorbed readily in the small intestine as flavin

nucleotides, i.e. FMN and FAD which is formed it intestinal mucosa.

Riboflavin is not stored to a considerable amount, although plasma,

erythrocytes, leukocytes, muscles, liver and kidneys contain riboflavin as

nucleotides, Riboflavin is secreted in the milk and in perspiration. It is

excreted mainly in the urine (0.1-0.4 mg. daily) and feces (500-750 g

daily). The urinary excretion of riboflavin increases with the increase in

dietary vitamin.

Biological function of riboflavin: Riboflavin is a component of two

important coenzymes: riboflavin mononucleotide (FMN) and riboflavin

adenine dinucleotide (FAD). The former is also known as riboflavin-5'-

phosphate and it combines with the various specific proteins (apoenzymes)

to form enzymes, viz. Warberg yellow enzyme. L-amino acid oxidase and

cytochrome-c-reeducates. On the other hand, FAD is composed of

riboflavin-5'-phosphate with a molecule of adenine-ribose-phosphate

(adenylic acid) and is thus a dinucleotide structure. If forms the prosthetic

group of a variety of enzymes, e.g. D-amino acid oxidase, xanthine oxidase,

aldehyde oxidase, glycine oxidase, etc. The structures of both of the

coenzymes have been established by means of analytical and synthetic

evidences.

452

TMN and FAD act as coenzymes in various H-transfer reactions in

metabolism. The hydrogen is transported by reversible reduction of the

coenzymes by two hydrogen atoms added to the 'N' at positions 1 and 10,

thus forming dihydro or leucoriboflavin. The principal enzyme reactions

catalyzed are as shown in Table 9.2.

Table 9.2

FMN FAD

Warburg's yellow enzyme

Cytochrome-C-reductase

L-amino acid oxidase

(Fp is autooxidizable at substrate level

by molecular O2 forming H2O2)

Xanthine oxidase

(Xanthineuric acid)

D-amino acid oxidase

Aldehyde oxidase

Fumarate dehydrogenase

(SuccinateFumarate)

Glycine oxidase

Acyl CoA dehydrogenase

Diaphorase

9.4.6 Co-Enzymes - Lipoic Acid (Thioctic Acid)

For a long time the unknown substance, responsible for growth of

protozoa and bacteria have been in active discussions some named these

substance as -Lipoic acid, while others called pyruvie oxidation species,

since this compound was found essential for oxidative decarboxylation in

strapto focus percales.

Later on it was found to be a sulphur containing fatty acid called 6, 8-

dithiooctanoic acid ( -lipoic acid or thioctic acid). It contains eight carbon

and two sulphur atoms. Oxidized and reduced forms of the compound is

shown below:

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It is a water-soluble material (difference from other members of the B

group) containing eight carbon and two sulphr atoms and hence is also

termed as thiocitc acid.

It is found is liver and yeast. It is a cofactor in the oxidative

decarboxylation of -keto acids e.g., pyruvate to form acetyl-CoA; and -

keto glutarate to form succinyl-CoA. In these reactions, lipoic acid functions

in conjunction with thiamine pyrophosphate (TPP) and serves as acyl-

generating, acyl-transferring, and a hydrogen-transferring agent.

Deficiency Manifestations: Not known lipoic acid occurs in a wide variety

of natural materials. Attempts to induce lipolic acid deficiency in animals

have so far been unsuccessful.

It is recognised as an essential component in metabolism although it is

active in extremely minute amounts.

As a coenzyme of Pyruvate dehydrogenase complex (PDH): It is

required alongwith other coenzyme in oxidative decarboxylation of

pyruvic acid to acetyl CoA.

As a coenzyme of -oxoglutarate dehydrogenase complex:

Required alongwith other coenzyme in oxidative decarboxylation of

-oxo-glutarate to succinyl CoA.

454

Lipoic acid is also required for the action of the enzyme sulfite

oxidase: Required for conversion of SO2- to So4

- - Hypoxanthine is

also required for the action.

9.4.7 Co-Enzymes - B212 (Cyanocobalamine)

Cobalamine has by far the most complicated structure of all the

vitamins. The ring structure resembles that of the porphyrins, but differ in

one respect that two of the pyrrole rings are joined directly rather than

through a methane group; this type of nucleus is known as the corrin

nucleus. The nitrogen atoms of the four substituted pyrrole rings surround

the cobalt atom in the centre and this structure is linked to a ribofuranose

residue to form cobamide. To this cobamide structure when a benzimidazole

nucleus is attached, cobalamine is produced. When the cobalt of cobalamine

is linked to -CN, cyanocobalamine is formed.

455

Cyanocobalamine is a deep-red, crystalline compound, soluble in

water. It is stable to heat in neutral solution but destroyed in acidic or

alkaline solutions.

When the cyanide group (anion) is replaced by-OH group the vitamin

is known as hydroxycobalamine, B12a. Similarly, cyanocobalamine is

converted into aquocobalamine, B12b and nitrocobalamine, B12c respectively.

Sources: Vitamin B12 of dietary importance is found in animal tissues. The

rich sources of vitamin are liver, egg, milk, meat and fish. Minute amounts

are probably present in all animal cells. Peculiarly, unlike other B vitamins,

B12 is not found in significant amounts in green plants and yeasts. Liver is

apparently an important storage site.

456

Vitamins B12 (extrinsic factor) is absorbed from the intestine only in

the presence of the intrinsic factor, a non-dialyzable, thermolabile substance

present in normal gastric juice and saliva, the precise nature of which is not

known but which has mucoprotein characteristics. It is secreted in milk, the

quantity increases with the increase in dietary vitamin. Although it is not

excreted in the urine under ordinary conditions and even when large

amounts (10 mg.) of it is ingested, it is excreted in the urine after

intravenous injections.

Functions: Like other vitamins of the B group, vitamin B12 is converted in

the living cell into a coenzyme known as coenzyme B12. In the conversion of

vitamin B12 to coenzyme B12, the cyanide group is replaced by a 5-

deoxyadenosine group. The coenzyme B12 is involved in the following

reactions.

(a) It is necessary for the conversion of methyl-malonyl-CoA to

succienyl-CoA by methylmalonyl-CoA isomerase.

Methylmalonic acid (MMA) can scarcely be detected in the urine of

healthy humans (less than 2 mg. per day), but its excretion in urine is greatly

increased (methylmalonic aciduria) in individuals with vitamin B12

deficiency such as in untreated patients with pernicilus anemia.

(b) It is also involved in the conversion of glutamate to -methyl

aspartate and diols to deoxyaldehydes, both of which occur in bacteria.

457

(c) A B12 containing protein is required for methylation of

homocysteine by 5-methyltetrahydrofolate to form methionine in animal

tissues. Note that this reaction represents one of the important functional

inter-relationship folic acid and vitamin B12.

(d) It is concerned in the protein biosynthesis.

(e) It is also involved in the metabolism of deoxyribosides and in

the conversion (reduction) of ribonucleotides to

deoxyribonucleotides.

As described in folic acid, vitamin B12 is involved in the formation of

red blood cells in the marrow (hemopoiesis). Actually, the deficiency in this

enzyme in man causes anti-pernicious anemia which is accompanied by

degradation of the spinal cord and thus vitamin B12 is known as anti-

pernicious anemia factor. The disease is characterized by a drastic decrease

of the erythrocyte count. Minute amounts (a few micrograms) of the vitamin

may cure pernicious anemia in man. It is important to note that the disease is

not always due to dietary deficiency of the vitamin (the extrinsic factor), but

rather is caused by a defect in the absorption of the vitamin owing to the

absence of the intrinsic factor (a mucoprotein in nature) formed in the gastric

juice. Most probably the intrinsic factor helps in liberating B12 from natural

protein complexes and in its subsequent transport in blood.

Cobalamine is a growth-promoting factor for a number of micro-

organisms and alges.

Check Your Progress - 1

Notes :(i) Write your answer in the space given below .

458

(ii) Compare your answer with those given at the end of the unit.

(a) (i) With the exception of..............................all the enzyme

are...................in nature, with.........................molecular weights.

Heloenzyme = ................................+........................

(ii)Co-enzymes are also known as..........................., and take part in

different reactions, such as (a).........................., (b)......................

and (c)........................and form..............bonds. They also

give.............................

(iii)The chemical names of coenzyme - A, B1, B6, B2 and B12

are......................

A - ...............................................

B - ...............................................

B6 - ...............................................

B2 - ...............................................

B12 - ...............................................

(iv)Nicotinic acid gives two cofactors:

(a) ..........................................., and

(b)...........................................;

while riboflavin gives (a)..................................................... and,

(b)................................................

459

9.5 ENZYME MODELS

Enzyme models are synthetic molecules which have one or more

properties of enzyme system. These have comparatively small and simple

structures than enzymes. That is in an enzyme model the principal

parameters of enzyme systems are of general nature and it comparatively

measure importance of each of the enzyme parameter. Enzyme molecules

are very complex and have a fix mechanism of actions. This fact caused the

necessity of enzyme-models. For planning a bio-organic enzyme model the

most important factor kept in mind is that how a limited number of common

functional groups, such as imidazole ring, aliphatic and aromatic hydroxyl

group, carboxyl group and amino groups, exhibit various reactions of known

enzyme system and calculate the enzyme reactions rate in their mechanism.

Generally any enzyme model has two main objectives;

(i) It follows a suitable enzyme mechanism, and

(ii) It explains the observed rate in terms of the structure and the steps

of its mechanism.

Thus an ideal enzyme model is one, which -

(a) provides best bonding site for the substrate, because non-covalent

interaction is the key of bioactivity specificity and sluggishness.

(b) has probability of electrostatic and hydrogen bonding, so that

substrate is suitably linked,

(c) selects catalytic groups carefully,

(d) has a stable and well defined structure, and

(e) is soluble in water and is catalytically active under the conditions

of pH and temperature.

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9.5.1 Host Guest Chemistry

D.J. Crome used the term 'Host-Guest Chemistry' for the use of crown

ethers in enzyme activity. In 1967 C.J. Paderson noticed that crown ethers

have ability to form stable complexes. They resemble with the ligands which

have cavity to bind the guest (metal or ammonium ions). Thus it has great

potential of being an active enzyme for a specific substrate.

Different shapes of cyclic polyethers with their crwon structures can

provide variable space for binding, due to their structure flexibility. These

compounds have simple molecules and can be prepared easily in different

sizes.

According to lock and key hypothesis enzymes play key role for

catalysis and inhibition or contraction during biochemical reactions. A high

structural complex molecule at least has one component of guest and a host

component. Host component is an organic molecule or ion, while the guest

component is in the form of a molecule or ion, which changes its binding

direction in the complex. Although, generally simple guests are available in

large number, but host are to be synthesized.

Host-Guest reaction represents stereoelectronic arrangement. The

synthetic host-guest complexes show polar and biopolar states. Micelles and

cyclodextrins are examples of natural hosts.

Similarly the prosthetic groups of hemoglobin, chlorophyll and

vitamin B12 may also be included in this group, as they remain bound with

iron, magnesium and cobalt ions respectively. Examples of some host

crowns are represented in Fig. 9.1

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Fig. 9.1

The host compounds shown in Fig. 9.1 are synthetic and represent

naphthalene as an example. These example show chirality of hosts with C2

symmetry.

9.5.2 Chirality and Catalysis

When any symmetry element is missing in the molecule, molecule

becomes asymmetric. The mirror and image configuration of such molecules

are not super impossible and hence called chiral molecules. This property of

substances is known as molecular chirality. Such compounds are optically

active and show optical isomerism. For example:

Chiral means hand, just as left and right hands do not super impose

like mirror and image. Chiral molecules give isomers which do not super

impose each other. Generally it has been seen S-host react readily but R are

sluggish, e.g. in the case of a complex formed with macro cyclic poly ether

462

host and amino ester salt as the guest. The transition state structure resulting

from S-host and L or D guest is shown in Fig. 9.2

Fig. 9.2

Molecular Recognition

Molecular recognition is the phenomenon which not only give

indication of bonding but also of selectivity and probable specific work. The

specific function to be done is related with the method of bonding which

give two different internally linked chemical levels. Out of these one

bioorganic specific level is formed by the accepted organic synthesis and is

used in identification to reach the second level.

These specific molecules have three different regions out of which

each one is responsible for a specific work and necessity. Thus one region is

concern with process of acceptable bonding used for recognition and is

necessary for high stability and selectivity. The second region is transporter

used for bonding and translocation necessary for loosening and transfer. It is

stable and involves fast change. The third region is a catalyst and is

concerned with bonding and transfer and used for molecular catalysis

necessary for high affinity and specific selection, for high reaction rate.

9.5.3 Molecular Asymmetry and Prochirality

463

The term prochirality was used by Prof. K.R. Hansen in 1966 to

define prochirality; a phenomenon in which replacement of a ligand from a

group, a chiral group is obtained. The original group is known as prochiral,

which consists of point ligand groups linked to prochiral centre.

Hansen proposed a successive process to lebel the face of trigonal

atom attached to CO group. The trigonal prochiral centre is planar in which

carbon atoms is surrounded by three principal groups a, b and c, These are

named according to R-S nomenclature:

S-Configuration

Enzyme catalysed biochemical reactions are sterospecific, hence

asymmetric synthesis are quite common in nature and are often necessarily

nondirectional. Most of the natural products are opticaly active e.g. enzyme

triosphosphate, by catalytic isomerisation converts achiryl dihydroxyacetone

monophosphate into D-glyceraldehyde phosphate:

Fructose Dihydroxy-acetone D-Glyceradohyde-

1-6 diphosphae mono-phosphate 3-phosphate

464

An other example is photosynthesis-phenomenon taking place in

plants, in which asymmetric synthesis converts solar energy into chemical

energy in the presence of chlorophyll. In this complex process achyril

carbondioxide converts into D-glucose.

In summary it can be said large number of natural products are chiral

substances, in which enzymes exert disymmetric effect. Other organic

substances are also help in biochemical processes.

9.5.4 Biomemetic Chemistry

'Biomemetic' term was introduced by Prof. R. Brasslo to show

chemical process taking place during a biochemical reaction using synthetic

models so that the structures and functions of complex salts can be known.

This would help in the study of mechanisms of enzymes with their

specificity.

Enzyme models are generally organic synthetic molecules which

share one or more properties of enzyme process. These molecules are

smaller and structurally simple as compared to enzyme molecules. Enzyme

models calculate comparative importance of each catalytic parameter. The

advantage of use of synthetic model structures for enzyme reactions is that

the model can be modified for the study of specific property. Thus

biomemetic chemistry, explores the region of excellaration and selective

properties of enzyme catalysed reactions. For this the molecules generally

used are (a) Crown ethers and Cryptases (b) Cyclodextrins (c)

Calixarenes (d) Ionosphere and (e) Micelles.

(a) Crown ethers and Cryptases

465

Generally alkali metals have very less tendency of complex formation,

however with organic compounds such as crown ethers they form stable

complexes in which they have co-ordination number 41 and 6. As a matter

of fact with monodentate ligands they do not form stable complexes, only

with bi-or polydentate ligands they form stable complexes.

The polyether complexes of alkali metals contain polycyclic ligand, in

the centre of which alkali metal atom is situated. These complexes have

crown shaped non-planar ring structures, e.g., dibenzo 18 Crown-6

potassium (I).

Dibenzo-18 crown-6 Potassium (I)

In the name of a complex the number before crown indicates the total

number of atoms present in the heterocycle, while the number after crown

gives the number of oxygen atoms. Thus in the above example 18 is the

number of atoms in the heterocycle and 6 represent the number of oxygen

atoms in the ring.

The most important property of crown ethers is that they make

inorganic salts soluble in non-polar organic solvents. Thus the specific

reactions which take place only in polar solvents, becomes possible in non-

polar solvents also. In these reactions crown ethers function as phase transfer

catalysts (Donald Crem, Charles Poderson and jeenmaria Leti were awarded

Nobel Prize for their work on crown ethers in 1987).

466

Naturally occurring ionospheres are also similar compounds in which

alkali metals are linked with oxygens of ethers, e.g., monactin valenomycin,

aniatin etc. which are antibiotic substances.

Cryptases

Macrocyclic crown compounds containing nitrogen or sulphur atoms

and macrocyclic compounds having more than one hetero atoms (N, S, O)

are also included amongst crown ethers and have similar properties.

Amongst these the bicyclic and polycyclic (tri, tetra cyclic) compounds are

called cryptods and their complexes are called cryptates. Tricyclic cryptod-8

has ten bonding sites and a cavity. It is known as spheroid. These polyethers

(cryptods) are used as models in biosystems. We know potassium is an

important enzyme activator. Sodium, inside muscles and potassium out side

muscles are important for their sensitivity and control.

(b) Cyclodextrins

Amylose is composed of the end to end linkage of glucose units. The

cyclic amylose with canal and cage structured complex are called

cyclodextrin's. The macro cycles containing 6, 7 or 8 glucose units are

nomad , and cyclo dextrin respectively. These are all soluble in water,

and the open space in them (, and ) are filled with 6, 12 and 17 water

467

molecules respectively using hydrogen bonding. The inside molecules are

less polar compared to outer one, which are quite polar.

1:1 cage complex of cyclodextrin is formed with many guests. If the

sizes of tricyclic dextrins are different, they can accommodate guests of

different sizes one over the other just as coins can be kept one over the other.

Cyclodextrins also form canal type complexes.

These complexes have catalytic activity, e.g. Prof. Brasslo and

Overman developed a model using cyclodextrin complex with a metal ion as

substrates. Thus, paranitrophenyl acetate substrate in pressure of Ni(II)

accelerate, rate of hydrolysis 1000 fold.

(c) Calixarenes

A new type of cyclic catalyst model was developed by Prof. C.O.

Gulse using benzene units. Due to its similarity with calix, it was named

calixarene. S.Shinbai developed a series of ligand and guests called 1, 3

alternate calix-4 arene. Calix (4) arene form inophoric cavity.

(d) Ionosphere

Ionospheres are natural chelating agents containing cyclic polyamides,

polyesters and polyethers, which have capability of selective binding with

metal ions.

For explaining permeability from natural or synthetic membrane

Kavanau divides a structural model. The inside non polar hydrophotic layer

of lipid and lipoproteins becomes permeable for a cation up to 10 nm

thickness.

468

In nature inophores are found in micro organism. Many of them are

used as antibiotic (e.g. valenomycin), bonded with metal ions they

catalytically regulate permeability of ions through bacterial membrane.

Valenomycin

(e) Micelles

In concentrated solutions of surface activators, due to hydrophobic

interaction, molecules cluster together to form colloidal particles in liquid-

crystal state called misclles (soap micelle is an important example, formed

during cleansing action of soap).

The surface of micelles is curved and have high cellular pressure,

hence the alkyl chain coil to give cluster of colloidal size. They may be

positively charged, negatively charged or neutral and may have spherical

lamellar or cylindrical shapes. They also play important part in bio-chemical

reactions, thus the contents of pancreatic juice are incapable of hydrolyzing

whole of the triglycerides, only 30-50% of it are hydrolysed in the intestine

and rest of the triglycerides, which remain undigested (un-hydrolysed) in the

intestine can be absorbed if it is dispersed in very fine particles, micelles.

This fine degree of dispersion of triglycerides is achieved by a combination

of triglycerides with bile salts, fatty acids and a monoglyceride. The

micelles, so formed can be absorbed actively (by some process) into the

microvilli. The monoglycosides and diglycerides (if any) absorbed in this

way are resynthesised into triglycerides, which are then transported by the

469

lymphatic circulation. Synthetic wetting agent Tween-80 (Sorbian

monooleate), having emulsifying action are used for promoting fat

absorption.

9.6 SYNTHETIC ENZYME OR SYN-ENZYME

An altogether new area of application of catalysts for industries is

emerging very fast. For this, synthetic molecules are prepared which have

catalytic activity similar to the natural enzymes. These synthetic enzymes

are called 'synzymes'. Before the adventure of synzymes, enzymes for

industries have been obtained from natural sources only.

Now it is not necessary that the starting material for synthetic

enzymes should be only protein. Synthetic enzymes are prepared from any

cyclodextrin, having property similar to trypsin. Such cyclodextins have 6 to

10 glucose units in cyclic form. In these molecules glycoside oxygen atoms

linked with CH radical form the hydrophobic environment, which works as a

bonding packet. The catalytic activity of these is developed due to linking of

imidazone, hydroxyl and carboxyl groups at the active centers. The catalytic

activity of these molecules is quite stable. Cyclodextrin protein is an

important synzyme. An another synzyme is methylmalonyl-CoA mutase

which is synthesised from methyl aspirate mutase with peptide-lipid

molecules.

Antibodies having enzymic properties have also been synthesised.

Check Your Progress - 2

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

470

(a) (i) Enzyme models have two main objectives:

(a) ..............................................

(b) ..............................................

(ii)Different shapes of cyclic polyether's with their............... can

provide......................for binding, due to their..............................

(iii)Host-guest relation represents..............................and

......................are examples of natural host.

(b) (i) Chiral means of.........................just as left and right hands do

not......................like mirror-image. Generally, S host

react..................., but R are.....................

(ii)Replacement of a..................................from a group. When

gives......................, the original group is known as.

(iii)Biomemetic chemistry involves use of synthetic enzyme models

to show........................taking place during.................. .

(iv) The molecule used in biomemetic chemistry are generally:

(a).......................................

(b).......................................

(c).......................................

(d).......................................

(e).......................................

9.7 LET US SUM UP

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After going through this unit you would have achieved the objective

given at the start of the unit. Let us recall what we have discussed so for:

With exception of ribosomes, all the enzymes are proteins in nature

with high molecular weights. Some enzymes are simple proteins

while some are conjugated proteins. In such enzymes the non-protein

part is called prosthetic group or co-enzyme and the protein part is

called apo-enzyme. The complete structure of apo-enzyme and

prosthetic groups or co-enzyme is called holo-enzyme:

Holoenzyme = Apoenzyme + Coenzyme

Coenzymes are also known as cofactors and take part in different

reactions such as redox, group transfer and isomerisation, and form

covalent bonds.

On the basis of their functions and the vitamins obtained from

them, cofactors are classified into different groups.

Co-enzyme may be defined as a substance necessary for the activity

of enzyme.

Pantothenic acid is a component of coenzymes-A (COA-SH) in turn

has a key role in metabolic processes, where its main function is the

transfer of acyl groups in both catabolic and biosynthetic reactions.

Thiamine pyrophosphate is coenzyme B. The physiological actions of

thiamine are due to its pyrophosphate (TPP), which together with -

Lipoic acid, is coenzyme of the enzyme carboxylases needed for the

decarboxylation of -keto acids such as pyrucvic and -ketoglutaric

acids.

472

Pyridoxal phosphate is coenzyme B6 which is involved in a number of

important metabolic reactions of the -amino acids, such as

transamination, recimization, decarboxylation and elemination

reactions.

Co-enzyme niacin has two active forms:

Nicotinamide Adenine dinucleotide (NAD) and

Nicotinamide Adenine Dinucleotide Phosphate (NADP).

These are the hydrogen transferring coenzymes. They accept

hydrogen from the substrate (which is oxidised) and then transfer step

by step to other compounds such as flavoproteins, cytochrome-b,

Cytochrome-c, Cytochrome-a and cytochrome oxidase. The overall

result is the oxidation of the substrate.

Co-enzyme B2 is riboflovin and has two active forms: Flavin

mononucleotide (FMN) and flavin adenine dinucleotide (FAD).

FMN and FAD acts as coenzymes in various H-transfer

reactions in metabolism.

Lipoic acid is responsible for growth of protozoa and bacteria. This

compound in essential for oxidative decarboxylation in straptofocus

phcaleas. It is required along with other co-enzyme in oxidative

decarboxylation of pyruvic acid into acctyl CoA.

Coenzyme B12 is cyanocobalamine, which is involved in (i)

conversion of methyl-malonyl CoA to succinyl CoA by methyl

malonyl CoA isomerase.

473

(ii) conversion of glutamate to -methyl aspartate and diols to

deoxyaldehyder, and

(iii) methylation of homocysteine by 5-methyl-tetra hydrofolate to

form methonine in animal tissues.

Cobablamine is a growth promoting factor for a number of micro

organism and algaes.

Enzymes models are synthetic molecules which have one or more

properties of enzyme systems, which have two main objectives:

(a) They follow suitable enzyme-mechanism, and

(b) Explain the observed rate in terms of the structure and the

steps of its mechanism.

Host-Guest chemistry term was used by D.J. Crome for the use of

crown-ethers in enzyme activity. According to lock and key

hypothesis they play key role for catalysis and inhibition or contrition

biochemical reactions.

Chiral molecules are optically active and show optical isomerism.

Generally it has been shown S-host react readily, but R are sluggish.

Molecular recognition in the phenomenon which not only give

indication of bonding but also of selectivity and probable specific

work.

The term prochiratiy is used to define the phenomenon in which

replacement of a ligane from a group gives a chiral group. The

original group is known as prochiral.

474

Enzyme catalyzed biochemical reactions are stereo specific, hence

asymmetric synthesis are quite common in nature and are often

necessarily non-directional.

Bio-mimetic chemistry in used to show chemical processes tacking

space during a biochemical reaction, using synthetic models. Enzymes

models are generally organic synthetic models which share one or

more properties of enzyme-processes. These molecules are

comparatively small and structurally simple than enzymes.

The molecules used for enzyme models are generally- (a) Crown-

ethers, (b) Cryptates, (c) Cyclodextrins (d) Calixarenes (e) lonophores

and (f) Micelles.

The synthetic molecules which have catalytic activity similar to a

natural enzyme are known as 'Synzymes' or 'Synthetic enzymes'. An

important class of compounds in this category is antibodies having

enzymic-properties.

9.8 CHECK YOUR PROGRESS: THE KEY

1 (a) (i) ribozymes

proteins

large

apo-enzyme + co-enzyme

(ii) Cofactors :

(a) redox (b) group transfer and (c) isomerisation

covalent

475

vitamins

(iii) A - Pantothenic acid

B1 - Thiamine Pyrophosphate

B6 - Pyridoxal Phosphate

B2 - Riboflavin

B12 - Cyanocobalamine

(iv) (a) Nicotinamide adenine dinucleotide (NAD)

(b) Nicotinamide adenine dinucleotide phosphate

(NADP)

Flavin mono nucleotide (FMN) and

Flavin adenine dinucleotide (FAD)

2. (a) (i) (a) It follows a suitable enzyme-mechanism.

(b) It explains the observed rate in terms of the

structure and the steps of its mechanism.

(ii) crown structure

variable space

structural flexibility

(iii) Stereo electronic arrangement

mecelle

cyclo dextrin

(b) (i) hands

super impose

readily

sluggish

(ii) ligand

chiral group

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prochiral

(iii) chemical process

biochemical reaction

(iv) (a) Crown ethers and cryptates

(b) Cyclodextrins

(c) Calix arenes

(d) Lonophore

(e) Micelle.

477

UNIT-10 BIOTECHNOLOGICAL APPLICATION OF ENZYMES

Structure

10.1 Introduction

10.2 Objectives

10.3 Large Scale Production and Purification of Enzymes

10.4 Immobilization of Enzymes

10.4.1 Techniques and Methods

10.4.2 Effect on Enzyme Activity

10.4.3 Applications

10.5 Uses of Enzymes

10.5.1 Food and Drinks (Brewing and Cheese Making)

10.5.2 Syrup and Corn starch

10.5.3 Drug Design and Enzyme Therapy

10.6 Enzymes and Recombinant Technology

10.7 Let Us Sum Up

10.8 Check Your Progress: The Key

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10.1 INTRODUCTION

Biotechnology has created revolution in modern age to change life of

human being. The new findings and experiments are giving specialised

information in different fields of life to make human more and more happy.

For long time enzymes have been used for the production of important

substances. For this enzymes have also been obtained from different sources

using different methods.

Several industrial processes such as food, dairy, sugar, soft drink,

brewery, textile, leather and pharmaceuticals use immobilized enzymes. The

immobilization moves fixing the enzyme and other molecule to or within

same suitable material. The objectives are to achieve as high an active

biocatalyst as possible.

The use of enzyme in drug design and in enzyme therapy has opened

new door for making human free from several diseases. Further, the giene

engineering using enzyme in recombinant of DNA technology has created

revolution in the field of treatment of diseases.

10.2 OBJECTIVES

The main aim of the unit is to study biotechnological applications of

enzyme. After going through this unit you would be able to:

describe large scale production and purification of enzymes,

discuss immobilization of enzymes and their applications,

analyse uses of enzymes, and

479

discuss recombinant technology of enzymes.

10.3 LARGE SCALE PRODUCTION AND PURIFICATION OF

ENZYMES

Enzyme may be divided under two groups, viz. inductive and

constitutive enzyme (Karstrom) on the basis of their formation. Inductive

enzymes are those whose formation is induced by some unphysiological

compounds (inducer), i.e. the organism is adapted to form these enzymes in

presence of certain unphysiological compounds called inducers and hence

they may also be known as adaptive. However, recent work has shown that

such enzymes are always produced by the organism, of course in small

amounts, but they may not be detected experimentally. The addition of' an

inducer accelerate the formation of such enzymes in detectable amounts. On

the other hand, the enzymes which are always produced by the organism

under normal physiological conditions are known as constitutive enzymes.

Quite related to induction there is another phenomenon involved in

the synthesis of enzymes. This process, known as depression, refers to

synthesis of enzymes in response to the absence of a specific small molecule

termed as co-repressor or negative inducer. In the phenomenon of negative

induction or regression the substrate (negative inducer) combines with

biosynthetic enzymes, responsible for formation of the relevant enzymes,

and renders them inactive and thus in case negative inducer is removed the

synthesis of the proper enzyme starts (depression). The phenomenon of

repression and depression have been demonstrated in Salmonella

typhimurium which can synthesise leucine (and hence enzymes involving)

leucine in the absence of externally added leucine (negative inducer). The

480

induction and depression phenomenon have principally been studied in

microorganisms.

Extraction of enzymes from sources and purification of extracted

enzymes is not easy. Extraction process selected on the basis of the nature of

source and on the basis of extraction process, the procedure of purification is

selected, so that maximum amount of the enzyme may be extracted and

purest form of it can be obtained.

For long time yeast has been used for industrial production of many

substances, hence it was thought microorganism may proved best source for

obtaining enzymes (Table 10.1).

Table 10.1: Formation of Some Enzymes

Organism Enzyme Inducer

Escherichia coli -Galactosidase Lactose

E. Coli Lysine decarboxylase Lysine

Streptococci Tyrosine decarboxylase Tyrosine

Bacillus cereus Penicillinase Penicillin

Pseudomonas Tryptophan peroxidase Tryptophan

Pseudomonas Formamidase Tryptophan

Pseudomonas Catechol oxidase Tryptophan

Animals Tryptophan pyrrolase Tryptophan

Animals Threonine dehydrase Threonine

Animals Tyrosine- -ketoglutaric

transaminase

Tyrosine

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Microorganisms are present in very large quantities hence variety of

enzymes may be obtained. For this it is not necessary that we take each

species of plant or organism, but based on genetic principles, according to

need for each specific reaction, specific enzyme is converted in to gene and

used for large scale production. This process is known as recombinant of

DNA technology. Using this technology not only different types of enzymes

may be obtained, but also in sufficient quantity. For examples, using

microbiotechnology, -galactosidase from e.coli and from asport carbomoil

transferi a new enzyme may be obtained in sufficient quantities

In addition, this may give different enzymes from eukaryotic type cell

using simple synthetic method and also revised and developed form of

enzyme may be obtained from revised gene.

Although animal and plant cells generally do not have capability of

production of enzymes on large quantities, but by enzymation and using

other revised microbacteria, from these cells also large quantities of specific

enzyme can be obtained and may be used for industrial production.

By controlling pH and temperature, culture of cells in suitable

medium, the growth of cells is increased many fold. The medium supplies

nutrition for the growth. After culture these are separated and extracts are

extracted on industrial level. The extract, are then separated in to

components on the basis of their nature using special separation methods.

After purification the enzyme of specific nature is obtained.

During enzymation reaction oxygen functions as a limited nutrient.

For increasing absorption capability, solution of sodium sulphite type

reducing agent is used with the enzyme. The reaction is carried out under

482

normal conditions. It is necessary that the medium of culture should be in

homogeneous state (in the order of PO2, OH, temperature and nutrient).

Mostly industrial or commercial enzymes are obtained by

fermentation hydrolyses process of microorganism (bacteria), which are

additional cellular enzymes and increase the quantity of enzyme. These

additional (extra) cellular enzymes are separated from cells by filtration or

centrifugation method. The continuous formulation process is used but it is

difficult to perform and control this process. Intracellular enzyme e.g.

glucose isomerage etc. are obtained from cells. However this does not give

the enzyme in large quantity. For this needle valve method at liquid top and

at high pressure is used. The remaining cell is separated from the centrifuge.

Thus, for large quantity extraction of enzyme continuous process

should be used and centrifuge. Machine is selected according to the size of

particles, while unwanted material is separated by filtration method.

Large Scale Purification of Enzymes

The enzyme obtained by above method is separated and concentrated

by microfiltration or evaporation method. For getting them free from toxic

substance specific purification methods are used. Which are selected on the

basis of nature of the enzyme molecule.

Using the parameters of enzymes, they are analysed for total reactive

protein concentration, conductivity, pH value and nucleic acid etc., and other

aprotic components. This information helps in deciding further procedure to

be adopted. For example if concentration of enzyme is weak then use of

microfiltration process becomes necessary. If total protein and nucleic acid

483

concentration is high, then viscosity of solution is reduced by dilution, or

nucleic acids are separated before chromatography.

Although there is no fixed process for purification but

chromatographic techniques are the best. Using this technique successively

large scale purification may be achieved.

10.4 IMMOBILIZATION OF ENZYMES

Immobilisation of an enzyme has been defined as the process by

which the movement of an enzyme in space is completely or severely

restricted, usually resulting in a water-insoluble from of the enzyme. The

process can be extended to cover cells and organelles. The immobilsation

can be achieved by fixing the enzyme and other molecules to or within some

suitable material. The carrier matrices are inert polymers or inorganic

materials used for immobilisation of enzyme. An ideal carrier matrix should

be economical, inert, stable and regenerable. In addition, it should increase

the enzyme specificity and reduce the production inhibition.

10.4.1 Techniques and Methods of Immobilization

Depending upon the nature of physical relationship of an enzyme to

the polymer matrix, the methods of immobilisation can be divided into

various groups. Thus the enzyme may be covalently bonded to the polymer,

physically adsorbed onto the polymer, cross linked with itself, entrapped

inside a carrier matrix or encapsulated in a 'polymer bag' (figure 10.1).

Moreover any combination of these methods can be applied to immobilize

the biological catalyst. In all the above mentioned methods, the polymer,

polymer matrix or carrier matrix employed for the effective immobilization

484

of the enzyme is in the form of material such as cellulose hydrogels, nylon,

glass, polyacrylamide beads or even iron filings. Despite the diversity of the

methods of immobilisation, the following four methods are generally used

for the same: (a) covalent bonding, (b) cross linking, (c) adsorption, (d)

entrapment, and (e) encapsulation.

(a) Covalent Bonding: In this method the catalysts are attached to the

polymer matrix by the formation of covalent bonds. This can be achieved by

two ways, the first by activating the polymer with a reactive group, the

second by the use of a bifunctional reagent to link an enzyme with the

polymer. Hydroxyl, amino groups or sulphydryl groups of an enzyme are

involved in covalent bonding with polymer molecule. Since the strength of

bonding is very strong it does not lead to loss of enzyme during use.

The large number of polymer matrices used in this process are

inorganic carriers such as ceramics, glass, iron, zirconium and titanium, the

natural polymer such as sepharose and cellulose, and the synthetic polymers

such as nylon, polyacrylamide and other vinyl polymer and copolymers.

The disadvantage of this technique is that the enzyme, may often be

inactivated due to conformational changes in the enzyme's active site which

can be overcome by performing immobilization in the presence of the

enzyme's substrate or a competitive inhibitor, or with proteases. Hydrogels

are the commonest polymer matrices that can be activated by the treatment

with cyanogens bromide to which biological catalysts bind to form

immobilized enzymes. The use of bifunctional reagents such as

gluteraldehyde that exist in the equilibrium mixture of monomers and

oligomers provides an alternative strategy for covalent enzyme

485

immobilisation. In addition to frequent inactivation of an enzyme, covalent

bonding has disadvantages in the form of use of toxic reagents, complicated

preparative procedures, and high costs involved.

(b) Cross-linking: The covalent bonding technique can be extended to the

cross-linking of the enzyme with the bifunctional reagent. In this approach,

bifunctional reagent molecules such as gluteraldehyde bind to two enzyme

molecules and a network of enzyme molecules linked together is produced.

The disadvantages of this method include loss of enzyme activity during

preparation, ineffectiveness for macromolecular substrates, and difficulty in

regeneration of carrier molecule. However, the technique is cheap, simple,

and widely used in commercial preparations of immobilised enzymes such

as glucose isomerase that is used in food industry.

(c) Adsorption: Perhaps this is the most widely used technique for

immobilisation of enzymes. It is the simplest and cheapest method where the

enzyme or cell adsorbs to a polymer material due to combination of

hydrophobic interactions and the formation of several salt links per enzyme

molecule. The method has additional advantages in that there is no

modification of enzyme and regeneration of carrier molecule is possible.

However, the technique has its own drawbacks, changes in ionic strength

may cause desorption and the enzyme is subjected to microbial or

proteolysis enzyme attack. The process requires careful selection of matrix.

The most commonly used matrices are, ion- exchange matrices, porous

carbon, clays, hydrous metal oxides, glasses and polymeric aromatic resins.

The technique involves most commonly employed adsorbant in the form of

ion exchange cellulose (such as DEAE cellulose), to which an enzyme is

mixed under appropriate conditions of pH and ionic strength. After

486

incubating this mixture for a sufficient period of time, the carrier is washed

to eliminate unabsorbed enzyme molecules, and the immobilized enzyme is

available for use.

Figure 10.1: Different methods of Immobilisation of enzyme

(d) Entrapment: In this method, catalysts are held or entrapped within

appropriate gels or fibres. The biocatalyst is dissolved in a solution of

polymer precursors and polymerisation initiated. The widely used polymers

are polyacrylamide gels, cellulose acetate, agar, gelatin or alginate.

Entrapment of an enzyme or microbial, animal and plant cells within

calcium alginate is the most widely employed method of immobilisation.

The technique has some disadvantages. Continuous loss of enzyme due to

variability of the pore size in gels, ineffectiveness in macromolecule

substrates and diffusion of substrate to the enzyme and of the product away

487

from the enzyme, make the preparation difficult and often result in enzyme

inactivation

(e) Encapsulation: This is a method in which the biocatalyst, usually in

an aqueous solution, may be enclosed in a semipermeable membrane

capsule. Encapsulation allows free movement in either directions to the

substrates and products but prevents the escape of biocatalyst from the

capsule. The technique is simple and cheap but an important condition is that

enzyme must be stable in solution. The biodegradable material in the form of

polylactic acid or phospholipid liposomes can be used for encapsulation of

enzyme. The major disadvantages of the technique is that the enzyme may

lose its stability and may undergo denaturation. The molecular weight of the

substrate is also influencing factor that minimises the applicability of this

technique.

10.4.2 Effect on Enzyme Activity

During the process of immobilisation of an enzyme, the objective is to

achieve as high an active biocatalyst loading per unit volume of

immobilisation support (polymer matrix) as possible. The different

parameters normally measured are the volume, enzyme activity, and protein

content or viable cell count of the enzyme or cell used, the weight, particle

size distribution, porosity and the chemical and physical properties of

polymer used, the activity and protein concentration of any biocatalyst

remaining after completion of immobilisation, operational stability, and

productivity and resistance to microbial contaminations.

Once the immobilisation of an enzyme is completed, the kinetic

behaviour of an immobilised enzyme differs significantly from that of its

488

free counterpart. Different enzymes show different response to the same

immobilisation technique. Therefore it is essential to employ a suitable

immobilisation technique for the given enzyme.

Immobilisation techniques may induce or suppress the stability of an

enzyme; it may put a strain in the enzyme molecule. The ultimate effect of

this strain is that the enzyme becomes vulnerable to denaturation by higher

temperatures, pH, etc. The other effects of immobilisation could include

total or partial inactivation caused by gross conformational change or

reaction of some essential group at the enzyme's active site, more subtle

induced conformational change causing destabilisation, alteration of

allosteric effects or kinetic characteristic stabilization. Limitation of the free

diffusion of solute molecules by the physical presence of polymer matrix

will cause the decrease in substrate molecules and concentration of the

product molecules around the enzyme. The pH 'profile' of an enzyme (i.e. a

graphical plot of the rate of enzyme activity versus pH) may be displaced,

distorted, broadened or narrowed as a consequence of immobilisation. An

electrostatic charge on the polymer matrix may have a direct effect on the

stability of ionizing groups at the enzyme's active site thus raising or

lowering their pKa value. In spite of these variations in enzyme kinetics, if

an enzyme molecule is subjected to multipoint binding, it may not create any

strain on it, which ultimately leads to substantial stabilisation.

10.4.3 Applications

Enzymes have an enormous range of applications in industry,

medicine, research, etc. The various uses of enzymes in solution are briefly

explained as follows:

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1. Biological detergents represent the largest industrial application of

enzymes. The chief enzymes utilised to manufacture detergents, soaps and

oxidants are as described below:

i. Proteases are used to digest proteins present in bloodstains, milk,

grass, dirt, etc. These enzymes are produced by Bacillus species.

The problem of allergic response of the workers and users which

was encountered before now is overcome by encapsulation

techniques.

ii. -amylase enzymes, produced by Bacillus species are used to digest

starch present in association with stains and/or dirt.

iii. Celluloses produced by fungi are used for washing cotton fabrics.

2. The baking industry uses the following enzymes

i. Fungal a-amylase enzymes catalyse breakdown of starch in the flour

to sugar, which can be used by the yeast. It is used in the production

of white bread, buns, rolls, etc.

ii. Proteinase enzymes are used in biscuit manufacture to lower the

protein level of the flour.

3. Brewing industry uses the following enzymes

i. and -amylases and -glucanase produced from barley; these

enzymes are utilised to degrade starch to produce simple sugars used

by the yeasts to enhance alcohol production.

ii. Amyloglucosidase and proteinases split polysaccharides and

proteins in the malt and use in production of low-calorie beer and

remove cloudiness during storage of beers.

4. Dairy industry utilises the following enzymes

490

i. Renin obtained from the stomach of young ruminant animals like

calves, lambs or kids and from fungus Mucor meihei is used in the

manufacture of cheese.

ii. Aspergillus derived lactase used to breakdown lactose to glucose

and galactose.

iii. Lipases produced by Rhizopus are employed for cheese ripening and

in ice-cream manufacture.

5. Textile industry uses the enzymes such as

i. and -amylases derived from Bacillus species are widely utilised

to remove starch which is used as desizing and binding agents on

threads of certain fabrics to prevent damage during weaving.

ii. Glucoamylases obtained from Aspergillus and Rhizopus are

generally preferred for desizing, since they are able to withstand

working temperatures upto 100-110oC.

6. Confectionary, soft drink and food industries is based on the

involvement of following enzymes-

i. Heat stable fungal proteases used for hydrolysis of gluten wheat,

make dough suitable for biscuit, pie and pastry making.

ii Amylases (- and -), glucoamylases, invertase and glucose

isomerases obtained from microbes act on starch, sucrose and D-

glucose to produce glucose, maltose arid high fructose syrups.

iii. Papain derived from papaya latex is utilised for tenderization of

meat.

iv Glucose oxidase derived from A. niger and Penicillium, catalyses

the formation of gluconic acid from - D glucose. The enzyme is

491

used for removal of glucose or oxygen from food in order to

increase its storability. In the process hydrogen peroxide (H2O2) is

produced which effectively kills bacteria.

v. Catalases derived from animal tissues are used to degrade H2O2 into

water and oxygen. It is usually applied in combination with glucose

oxidase to remove glucose and/or O2, from foods, drinks, etc.

vi. Pectinases and cellulases obtained from Aspergillus are used to

reduce viscosity and increase juice yield with enhanced flavour.

7. Leather Industry

Traditionally enzymes found in dog and pigeon faeces were used to

treat leather to make it pliable by removing certain protein components the

process is called bating; strong bating required to achieve a soft, pliable

leather, slight bating for the soles of shoes. Bating is also essential for

production of soft leather clothing. Nowadays trypsin an enzyme obtained

from slaughterhouses and from microorganisms is utilised for removing the

hair from hides and skins to increase suppleness and softness in appearance.

8. Pharmaceuticals and medical industries

The trypsin, obtained as mentioned above, is used in debridement of

wounds, dissolving blood clots. The pancreatic trypsin is utilised for

digestive aid formulations and treatment of inflammations. Many enzymes

are used in clinical chemistry as diagnostic tools some of which are

employed in treatment of diseases.

Chemical industry utilises immobilised nitrilase to produce

acrylamide from acrylnitrile. The enzyme nitrile hydratase (or nitrilase) is

obtained from Rhodococcus.

492

9. Biosensor

A biosensor is an analytical device that involves the combination of

biological active material displaying characteristic, specificity with chemical

or electronic sensor to convert a. biological compound into an electrical

signal. These electric signals are amplified, interpreted and displayed to

measure the concentration of compound present in the solution.

In design, the enzyme electrode or biosensor is composed of a given

electrochemical sensor in close contact with a thin permeable enzyme

membrane capable of reacting specifically with the given substrates. The

embedded enzymes in the membrane produce O2 hydrogen ions, ammonium

ions, CO2, heat, light or even directly electrons depending upon the enzymic

reaction occurring, which are readily detected by the specific sensor. The

magnitude of the response determines the concentration of the substrate.

Sensor can measure a reduction in one of the substrates or an increase in one

of the products of the reaction catalysed by the biosensor. Although the

biological component in a biosensor may more often be an enzyme or multi-

enzyme system, it can also be an antibody, nucleic acid, an organelle, a

microbial cell, and whole slices of tissue or entire organs.

Considering an example of a simple glucose electrode which can be

constructed by immobilising a layer of glucose oxidase, in polyacrylamide

gel around a platinum oxygen electrode, the concept can be made clear.

When a solution of glucose is brought into contact with the electrode,

glucose and O2 diffuse into the enzyme layer and are converted to

gluconolactone and H2O2, lowering the O2 concentration in the gel layer

around the electrode. The rate of reaction is recorded as the rate of depleting

O2 concentration. Such a device responds linearly to glucose concentration

493

over a range of 10-1

-10-5

mol dm-3

with a typical response time of 1 minute

and is stable for up to 4 months.

The biosensor is of various types depending on the physical changes

that occur in the vicinity of the sensor. These physical changes may be (i)

heat released or absorbed by the reaction that can be measured by

calorimetric biosensors, (ii) production of an electric potential due to altered

distribution of electrons that can be detected by potentiometric biosensors,

(iii) movement of electrons due to redox reaction which can be measured by

amperometric biosensors, (iv) light produced or absorbed during reaction

that is detected by optical biosensors, or (v) change in mass of biological

component as a result of reaction, which can be measured by acoustic wave

biosensors.

Biosensors have tremendous role to play in world economy. The

estimated world market is approximately $ 25 billion/year of which 30% is

in healthcare. At present only 0.1% of this market is using biosensors.

Biosensors have been constructed to measure almost anything from blood

glucose level to the freshness of fish. Looking to the future, we can expect

that biosensors will play an indispensable role in detecting pollutants present

in the environment. Biosensor market is likely to flourish with the

development of economical, easy-to-use and more stable biosensors.

Check Your Progress - 1

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

494

(a) (i) Biotechnology has created..............................in modern age to

change...........................of human being. Enzyme....................... has

opened new door for making human........................

several...................

(ii)Micro organism have proved........................for

obtaining..................Enzyme in large quantity are obtained

using...........................technology.

(iii)Best method for large scale purification of enzyme is.....................

(b) (i) Immobilization of an enzyme is the process by which

....................of an enzyme in......................is completely

...................It can be achieved by........................the enzyme

and.....................to or....................some suitable material.

(ii) The methods used for immobilisation are:

(a) ............................................

(b) ............................................

(c) ............................................

(d) ............................................

(e) ............................................

(ii) Application of immobilized enzyme are in (a)..................,

(b)........................, (c)......................., (d)......................,

(e)........................ and (f)......................industries.

10.5 USE OF ENZYMES

495

In spite of the disadvantages mentioned in various techniques, the

immobilised enzymes have a number of advantages over the free

biocatalysts.

1. Immobilised enzymes are normally more stable than their soluble

counterparts.

2. They can be reused in the purified, semi-purified or whole-cell form.

3. Catalytic properties of immobilised enzymes can be altered favorably

and this permits the enzyme to function under broader or more

rigorous reaction conditions; increase in thermostability is one such

favorable change in immobilised enzymes e.g. An immobilised

glucose isomerase can be used continuously for over 1000 hours at

temperatures between 60 and 65oC, while its normal form denatures at

45oC.

4. Since the product can be readily separated from the enzyme, it

effectively saves the cost of downstream processing of the product.

5. Immobilisation of an enzyme makes recovery easy and may also

reduce effluent-disposal problems.

6. It allows more accurate control of catalytic processes.

7. It provides an ideal system for continuous operation.

8. It offers considerable potential for industrial and medical use.

Several industrial processes are based on the use of immobilised

enzymes, as it is advantageous than enzymes in solution. Some of the

common industrial applications of immobilised enzymes are as follows.

10.5.1 Food and Drinks (Brewing and Cheese Making)

496

Food industry and drinks industry utilises immobilised glucose

isomerase from Actinoplanes missouriensis, Bacillus coagulans and

Streptomycessp. This enzyme is used to convert glucose syrup into high

fructose syrup, which is exploited for preparation of soft drinks, since

fructose is sweeter than glucose. Immobilised aminoacylase is another

enzyme employed for resolution of D, L- ammo acids to L-form.

In dairy industry immobilised lactase is used to hydrolyse lactose to

glucose and galactose. Since many people are sensitive to lactose, lactase is

used to remove the lactose from milk and whey.

10.5.2 Syrup and Corn Starch Industry

A Sugar industry utilises immobilised raffinase to digest raffinose

present in sugar beet juice; this helps to increase sugar recovery by 3% and

reduces the cost of disposal of molasses. Raffinase can be isolated from the

mould Mortierella vinacea. Sugar industry also exploits immobilised

invertase to produce invert sugar. For applications in syrup industry see

section 10.4.3.

10.5.3 Drug Design and Enzyme Therapy

Pharmaceutical industry involves the use of immobilised penicillin

amidase to synthesise penicillin and cephalosporin-the antibiotics exploited

commercially to combat bacterial diseases. (Also see section 10.4.3) e.g.

i. -Amylases, lipases, proteases are used in treatment of digestive

disorders.

497

ii. Asparagines, glutamines are utilised for treatment of cancer,

specifically leukemia,

iii. Ribonuclease is used as antiviral agent.

iv. Rhodonase is used for cyanide poisoning.

v. -lactamase is used in treatment of penicillin allergy.

vi. Streptokinase and urokinase for treatment of the disorders of blood

circulation.

Researches believe that chemical genetics is a step closer to drug

development. In the last decade the rapid development of bacterial resistance

to antibiotics has generated a critical medical need for new therapeutics.

Scientists, have found the effect of small molecules are generally fast,

reversible, tunable, easily accessible, can be initiated and studied at different

stages in the development of the organism. The identification of novel

molecular targets and small molecules for the treatment autoimmune

diseases and cancer has been reported e.g. researchers have discovered a

new enzyme GAPDH, which regulates insulin pathways. This finding

offered a new direction for the treatment of diabetes. They used the worm

cacnorhaditis elegans to identify a new therapeutic target protein for diabetic

treatment.

Thus, the activity of enzymes indicated that enzymes play important

part in the diagnosis and treatment of many diseases and this medical tool is

known as clinical enzymology.

Clinical significance of enzyme assay have been proved quite useful

in many diseases, when the normal tests fail to give conclusive results. Thus-

498

(a) In heart disease following serum enzymes assay give important

information :

(i) Creative phosphokinase (CPK or CK).

(ii) Serum Glutamate Oxaloacetate transaminase (S-GOT).

(iii) Lactate dehydrogenase (LDH).

(iv) r-Glutamyl transpeptidase (G-GTP)

(v) Histaminsae

(vi) Cholinesterase

(b) In liver diseases

(i) Serum anylase and serum lipase

(c) In muscle diseases

(i) S-GOT/S-GPT (ii) Serum aldolase, and (iii) Serum CPK

(d) In bore diseases

(i) Acid phosphotase and (iii) -Glucuronidase

Table 10.1 gives increase/decrease of different enzyme in diseases:

S.

No.

Serum

enzymes

Normal value Concentrations increased in Concentration

s decreased in

1. Aspartate

transaminase

(AS-T)

(S-GOT)

4-17 IU/L Myocardial infarction, elevation

slight to moderate in muscle

diseases, acute liver disease,

toxic liver cells necrosis,

haemolytic anaemia.

--

2. Alanine

transaminase

(ALT)

3-15 IU/L Marked increase : viral

hepatitis,

Slight to moderate-obstructive

499

S.

No.

Serum

enzymes

Normal value Concentrations increased in Concentration

s decreased in

(S-GPT) jaundice, cirrhosis liver, toxic

liver cells necrosis, skeletal

muscle disease.

3. Lactate

dehydrogenas

e (LDH)

60 to 250 IU/L Acute myocardial infarction,

acute hepatitis, also raised in

muscle diseases, leukaemias,

renal tubular necrosis,

carcinomatosis, cerebral

infraction,pernicious anaemia.

4. Alkaline

phosphatase

(ALP)

3 to 13 K.A.

units % (23-92

IU/L) Infants

and growing

children 12-30

K.A. units per

100 ml.

Marked increase : obstructive

jaundice (> 35 K.A. units%),

bone diseases –rickets, Paget's

disease, hyperparathyroidism.

Slight to moderate increase :

acute liver diseases, metastatic

carcinoma, "space-occupying"

lesions of liver, kidney

disease, osteoblastic sarcoma.

5. Creatinine

kinase (CK

or CPK)

4-60 IU/ L Marked increase: acute

myocardial infarction,

muscular dystrophies;

Mild to moderate rise : muscle

injury, severe physical

exertion, hypothyroidism.

6. Aldolase 2 to 6 m-IU Muscular dystrophies, acute liver

diseases, myocardial infarction,

diabetes mellitus, leukaemias etc.

7. Amylase 80 to 180

Somogyi units

%

Acute pancreasitic, acute parotitis

(mumps), perforated peptic ulcer,

intestinal obstruction, macro-

amylasemia, renal failure

Acute liver

diseases,

D. mellitus

8. Lipase * Colorimetric

assay 9.0 to 20

Acute pancreasitic, perforated

peptic ulcer, cirrhosis liver,

Acute liver

diseases.

500

S.

No.

Serum

enzymes

Normal value Concentrations increased in Concentration

s decreased in

m-IU

(Seligman and

Nachlas)

* Titrimetric

method 0.06 to

1.02 ml. of

0.05 (N)

NaOH.

*Cherycrandal

units. 1.0 to

1.5 units %

Pancreatic carcinoma D. mellitus,

vitamin A

deficiency.

9. Cholinesterase 2.17 to 5.17

IU / ml, 130-

310 units (dela

Huerga)

Nephrotic syndrome, acute

myocardial infarction

Acute liver

diseases,

Malnutrition,

acute

infectious

diseases,

organo-

phosphorous

poisoning

(diazinon

poisoning)

10. Acid

phosphatase

(ACP)

0.6 to 3.1 KA

units / 100 ml.

0 to 0.08 KA

units %

Metastasizing prostatic

carcinomas, marked rise seen in

Gaucher's disease.

Slight to moderate rise seen in

Paget's disease, hyperpara-

thyroidism, osteolytic lesions

from breast carcinoma,

thrombocytosis

Slight increase after rectal

examination (P.R.), chronic

granulocytic leukaemia,

myeloproliferative lesions.

501

S.

No.

Serum

enzymes

Normal value Concentrations increased in Concentration

s decreased in

11. Ceruloplasmin

(Ferroxidase)

3 to 58 mg % Cirrhosis, bacterial infections,

pregnancy

Wilson's

disease

(hepatolenticu

lar

degeneration)

12. Isocitrate

dehydrogenase

(ICD)

0.9 to 4.0 IU/L Marked increase seen in viral

hepatitis.

Slight to moderate rise

cirrhosis liver.

13. Ornithine

carbamoyl

transferase

(OCT)

8 to 20 m-IU Marked elevation in viral

hepatitis;

Slight elevation-cirrhosis

liver, obstructive jaundice

metastatic carcinoma.

--

14. Leucine

amino-

peptidase

(LAP)

15 to 56 m-IU Marked rise in-liver cell

carcinoma

Slight increase-cirrhosis liver,

marked rise in superimposed

hepatoma in cirrhosis liver,

Moderate rise, viral hepatitis.

15. -Glutamyl

transpeptidas

e (-GT)

10 to 47 IU / L Acute hepatobiliary diseases,

alcohol abuse marked rise

characteristic, alcoholic cirrhosis,

slight to moderate increase seen

in epileptic patients with drug

therapy with anticonvulsants,

pancreatic diseases.

-

16. 5'-

Nucleotidase

2 to 17 IU/L Acute liver diseases, obstructive

jaundice, tumours.

--

10.6 ENZYMES AND RECOMBINANT TECHNOLOGY

This technology is concerned with the transplantation of gene from

one organism to the other. This depends primarily on enzymes, as enzymes

502

only can break DNA-chain into specific parts and can manipulate. New

portion of DNA are then joined at the place where the enzyme has broken

the DNA chain and the chain is repaired. When the transplantation becomes

successful it is tested for the result. Thus the technique is based on enzyme

and involves alteration in DNA in an organ to produce the required protein.

The protein thus produces becomes useful in human treatment for a

particular disease. For example pig insulin resemble with human insulin.

Human insulin produced by bacteria is now available in the market for the

treatment of diabetes patients. In this technique the steps involved are-

(i) study of DNA structure, properties and uses,

(ii) use of specific enzyme for dissociation of DNA,

(iii) sealing of DNA portion in the cavity, and

(iv) Generation of revised DNA

Check Your Progress - 2

Notes :(i) Write your answer in the space given below .

(ii) Compare your answer with those given at the end of the unit.

(a) (i) Researchers believe that ..................................... is a step closer

to....................................

(ii)Enzymes play important part in the......................and..............of

many diseases. This........................is known as......................

(iii)Enzyme assay give.............................information

about...............Thus, ..................and...................in liver diseases.

(iv)Recombinant technology is based on.............. & involves........

in.................................in an organ to produce the.........................

503

10.7 LET US SUM UP

By going through this unit your would have achieved the objective

given at the start of the unit. Let us recall what we have discussed so for:

Enzymes may be divided into two groups: Inductive and constitutive

enzymes. Inductive enzymes are those whose formation is induced by

some physiological compounds (inducer). On the other hand, the

enzymes which are always produced by the organism under normal

conditions are known as constitutive enzymes.

Extraction of enzymes from sources and purification of extracted

enzyme is not easy. Extraction process is sleeted on the basis of nature

of source and on the basis of extraction process, the process of

purification is selected so that maximum amount of the enzyme is

extracted and in purest from it can be obtained.

Based on genetic principles, according to need for each specific

reaction specific enzyme is converted into gene and used for large

scale production. This process is known as recombinant of DNA

technology.

Although animal and plant cells generally do not have capability of

producing enzymes in large quantities, but by enzymation and using

other revised micro bacteria, large quantities of specific enzyme can

be obtained from these cells also, may be used for industrial

production.

Under suitable medium and conditions the growth of cells is increase

many fold in the culture. After separation from the culture, extracts

are obtained and separated in to components using special separation

504

methods based on their nature. After purification specific enzymes are

obtained.

Mostly, industrially/commercially enzymes are obtained by

fermentation hydrolases process of micro-organism, which are

additional cellular enzymes and increase the quantity of enzyme.

These additional enzymes are separated by filtration or centrifugation

method.

For large quantity extraction of enzymes continuous process is used.

Enzyme obtained by above process are separated and concentrated by

micro-filtration or evaporation method. For getting them free from

toxic substances specific purification methods are used; which are

selected on the basis of nature of the enzyme molecules. Mostly

chromatographic techniques are proved amongst the best purification

methods.

Immobilisation of enzyme is the process by which the movement of

an enzyme in space is completely or severely restricted.

Depending upon the nature or physical relationship of an enzyme to

the polymer matrix, the methods of immobilisation can be divided

into various groups. Despite the diversity of the method, the following

four methods are generally used: (a) Covalent bonding, (b) Cross

linking, (c) Adsorption, (d) Entrapment and (e) Encapsulation.

During the process of immobilisation of an enzyme, the objective is to

achieve as high an active biocatalyst loading per unit volume of

immobilisation support as possible.

505

Once the immobilisatin of an enzyme is completed, the kinetic

behaviour of an immobilised enzyme differs significantly from that of

its free counterpart.

Enzymes have enormous range of application in industry, medicine

and research. Their industrial application include, food and drinks,

syrup and corn starch, backing, brewery, dairy, textile, leather and

pharmaceutical industries.

They are also used to prepare biosensor and play important part

in drug design and enzyme therapy. Biotechnology involving gene

engineering for recombinant DNA technique has revolutionized the

method of treatment of many dangerous human diseases.

10.8 CHECK YOUR PROGRESS: THE KEY

1 (a) (i) revolution

life

therapy

free from

diseases

(ii) best source

enzymes

recombinant DNA

(iii) chromatographic technique

(b) (i) movement

space

restricted

fixing

506

other molecules

within

(ii) (a) Covalent bonding

(b) Cross linking

(c) Adsorption

(d) Entrapment, and

(e) Encapsulation

(iii) (a) Detergent

(b) Food and Backing

(c) Soft drinks and brewery

(d) Textile

(e) Leather

(f) Pharmaceutical Industries

2 (i) chemical genetics

drug development

(ii) diagnosis

treatment

medical tool

clinical enzymology

(iii) conclusive

diseases

serum amylase and

serum lypase

(iv) enzyme

alteration

DNA

required protein