PROTEINSCOLLEGE OF MEDICAL TECHNOLOGY

LECTURE

OBJECTIVES

• The students would be able :

• 1. to describe and recognize amino acids structures and illustrate how they are connected in

proteins.

• 2. to describe how the properties of amino acids depend on their side chains and how their

ionic charges vary with pH.

• 3. to define protein primary structure, explain how primary structures are represented, and

draw a simple protein structure, given its amino acid sequence.

• 4. to explain what is responsible for handedness and recognize simple molecules that display

this property.

• 5. to describe and recognize disulfide bonds, hydrogen bonding along the protein

backbone, and noncovalent interactions between amino acid side chains in proteins.

• 6. to be able to define these structures and the attractive forces that determine their

nature , describe the αlpha helix and β-sheet, and distinguish between fibrous and

globular proteins.

• 7.to be able to define quarternary structures and give examples .

• 8. to describe protein hydrolysis and denaturation , and give some examples of

agents that cause denaturation.

• Biochemical reactions must continuously break down food molecules, generate

energy and store energy, build up new molecules, and eliminate waste.

• Each biomolecule has a role to play in these processes.

• All the principles of chemistry introduced thus far apply to biochemistry.

• Functional Groups are of greatest importance in biomolecules.

PROTEINS

• Large biological molecules made of many amino acids linked together

through amide (peptide) bonds.

• Two or more amino acids can link together by forming amide bonds.

• Dipeptide results from the formation of a peptide bond between the –

NH2 group of one amino acid and the – COOH group of a second

amino acid.

FUNCTIONAL GROUPS

• Amino group -NH3 ; -NH2

• Hydroxyl group -OH

• Carbonyl group - C=O

• Carboxyl group - COOH

• Amide group - CN=O

• Carboxylic acid ester - C=OOR

• Phosphates PO2 ;PO3;

• Hemiacetal group - C-OH Acetal - C -OR

l l

OR OR

DEFINITIONS:

• Amino acid – molecule that contains both an amino group and a carboxylic

acid group

• Alpha – amino acid – amino acid in which the amino group is bonded to the C

atom next to the –COOH group.

• Side chain – group bonded to the C next to the carboxyl group in an amino

acid

• Zwitterion – a neutral dipolar ion that has one + charge and – charge.

POLYMERS OF AMINO ACIDS

• Every amino acid in a protein contains an amine group(NH2), a

carboxyl group, and an R group.

• R groups may be hydrocarbons, or may contain a functional group.

• Alpha amino acids – the amine group is connected to a C atom

connected to the carboxylic acid group.

• Peptide bond – an amide bond that links two amino acids together.

• Intermolecular forces are of central importance in determining the

shape and functions of proteins.

• Covalent – act between organic compounds

• Noncovalent – act between different molecules or between

different parts of the same large molecule

• Hydrophobic – water – fearing; a hydrophobic substance does not

dissolve in water

• Hydrophilic – water – loving; a hydrophilic substance dissolves in

water

• Isoelectric point – the pH at which a sample of an amino acid has

equal numbers of + and - charges

AMINO ACIDS

• Non polar side chain

• Polar, neutral Side chains

• Acidic Side chains

• Basic Side chains

NONPOLAR SIDE CHAINS

• Alanine

• Glycine

• Isoleucine

• Leucine

• Methionine

• Phenylalanine

• Proline

• Tryptophan

• Valine

POLAR, NEUTRAL SIDE CHAINS

• Asparagine

• Cysteine

•Glutamine

• Serine

• Threonine

• Tyrosine

ACIDIC SIDE CHAINS

• Aspartic acid

•Glutamic acid

BASIC SIDE CHAINS

• Arginine

• Lysine

• Histidine

AMINO ACIDS: NATURE

• Nature uses the 20 alpha – amino acids to build the proteins in all

living organisms.

• 19 of them differ only in the identity of the R group

• Classified according to their acidity and basicity.

• Classified as nonpolar side chains and polar side chains.(15 )

PROPERTIES OF AMINO ACIDS

• Amino acid contain both an acidic group, COOH, and a

basic group, -NH2.

• These groups can undergo intramolecular acid-base

reactions.

• Dipolar ions are known as zwitterions, which allows many

physical properties (salts).

• Can form crystals . ( pure form)

• Have high melting points.

• Soluble in water

• Insoluble in organic solvents

ESSENTIAL /NONESSENTIAL AMINO ACIDS

•ESSENTIAL AMINO ACIDS - Cannot be synthesized by the

body and must be obtained from the diet

•NON ESSENTIAL AMINO ACIDS – can be synthesized by

the body

TWO NEW AMINO ACIDS

• Selenocysteine – (Sec) ;21st amino acid; it is not coded directly in the genetic

code

Sec – encoded by a UGA codon, stop codon; has a specialized tRNA;

present in formate dehydrogenase, glycine reductase

• Pyrrolysine – (Pyl) ; 22nd amino acid; genetically encoded used by archaea in

enzymes that are part of their methane-producing metabolism

AMINOACIDOPATHIES

• Class of inherited errors of metabolism in which there is an enzyme defect that inhibits the body’s ability to

metabolize certain amino acids

• Exist : 1. in the activity of specific enzyme in the metabolic pathway

2. in the membrane transport system for amino acids

Phenylketonuria

Tyrosinemia

Alkaptonuria

MSUD – Maple Syrup Urine Disease

Other Examples of Aminoacidurias:

Isovaleric Acidemia

Homocystinuria

Citrullinemia

Arginosuccinic Aciduria

Cystinuria

HANDEDNESS

• Chiral – having right or left handedness; able to have two

different mirror – image forms

•Mirror image – If you hold your left hand up to a mirror, the

image you see looks like your right hand.

• Superimposable – It is easy to mentally place the chair on

top of its mirror image.

• Achiral – having no right or left –handedness and no non

superimposable mirror image

CHIRALITY

• Alanine – chiral molecule

• Its mirror images cannot be superimposed on each other.

• Result: it exist in two different forms that are mirror images

of each other.

• D- alanine & L-alanine ( enantiomers or optical isomers )

• Propane – achiral molecules

• Stereoisomers – compounds with same formula and atoms

with same connections but different spatial arrangements

LEVELS OF STRUCTURE

•Primary

•Secondary

•Tertiary

•Quarternary

LEVELS OF STRUCTURE: PROTEINS

• Primary structure -Sequence of amino acids in a protein chain

• Secondary structure – Regular and repeating spatial organization of

neighboring segments of protein chains

• Tertiary structure – Overall shape of a protein molecule produced by regions

of secondary structure combined with the overall bending and folding of the

protein chain

• Quarternary structure – Overall structure of proteins composed of more than

one polypeptide chain

PRIMARY STRUCTURE

• Sequence in which its amino acids are lined up and connected by

peptide bonds.

• Backbone of protein is a chain of alternating peptide bonds.

• Amino – terminal amino acid – free –NH3 group at the end of a protein

• Carboxyl- terminal amino acid – free carboxyl group at the end of a

protein

• Zwitterions give amino acids many physical properties.

• Pure amino acids :

•A. can form crystals.

• B. high melting points

•C. Soluble in water

•D. Insoluble in non polar solvents

CHARGE OF AMINO ACIDS

• Depends on the following:

1. Structure of amino acids

2. pH of medium

Isoelectric point – pH at which the net positive and negative charges

are evenly balanced (pI)

* At this point , the overall charge of all the amino acids in

sample is zero.

INTERACTION: DETERMINE THE SHAPE

• Structure – function relationship , depends on the polypeptide chain

being held

1. Hydrogen bonds along the backbone

2. Hydrogen bonds of R groups with each other or with Backbone atoms

3. Ionic attractions between R groups

4. Hydrophobic interactions between R groups

5. Covalent Sulfur-Sulfur bonds

SECONDARY STRUCTURE

• Includes two repeating patterns:

a. alpha – helix

b. Beta – sheets

Hydrogen bonding between backbone atoms holds the polypeptide chain in place

Connects the carbonyl oxygen atom of one peptide unit with the amide hydrogen of

another peptide unit

The repeating patterns arranged in loops and coils in random

ALPHA - HELIX

• Single protein chain coiled in a spiral with a right – handed (clockwise)

twist

• Resembles a phone cord, stabilize by hydrogen bonds between peptide

groups along its backbone

• Secondary structure

BETA SHEET

• Protein chains in the same or different molecules

• Secondary structure

• Held in place by hydrogen bonds along the backbones

• Stack pleated sheets allow R groups above and below the sheets

SECONDARY : FIBROUS AND GLOBULAR

• Fibrous proteins – tough, insoluble; wool, hair, fingernails –alpha

keratins

• Hardness, flexibility, and stretchiness varies with the number of

disulfide bonds

• Composed of alpha – helices

• Examples: Keratins, Elastin , Myosin, Fibrin

• Keratins – found in skin, wool, feathers, hooves, silk, fingernails

• Collagen – found in animal hide, tendons, bone, eye cores, and

connective tissues

•Myosins – found in muscle tissues

• Fibrin – found in blood clots

•Globular proteins – water – soluble protein whose chain is

folded in a compact shape with hydrophilic groups on the

outside

•Water loving outside accounts for their side chains

• Tertiary structure

• Examples: Insulin, Ribonuclease, Immunoglobulins,

Hemoglobin, Albumins

• Ribonuclease – enzyme that catalyzes RNA hydrolysis

• Insulin – regulatory hormone for controlling glucose

metabolism

• Immunoglobulins – proteins involved in immune response

• Hemoglobin – protein involved in oxygen transport

• Albumins – proteins that perform many transport functions in

blood; protein in egg white

TERTIARY STRUCTURE

• over-all three dimensional shape that results from the

folding of a protein chain

• Depends mainly on interactions of amino acid side chains

that are far apart along the same backbone.

• Noncovalent interactions and disulfide covalent bonds

• Native protein – with the shape (secondary, tertiary,

quarternary) in which it exists naturally in living organisms

CONJUGATED PROTEINS

• Glycoproteins – CHO; found in cell membranes

• Lipoproteins – Lipid; HDL,LDL,VLDL, chylomicrons

• Metalloproteins – Metal ions; cytochrome oxidase

• Phosphoproteins – Phosphate groups; milk casein

• Hemoproteins – Heme ; Hemoglobin, myoglobin

• Nucleoproteins – RNA; found in cell ribosomes

QUARTERNARY STRUCTURE

• Final level of protein structure; most complex

• Two or more polypeptide subunits form a single-three

dimensional protein unit

• Held together by noncovalent forces and covalent forces

• Hemoglobin – composed of four polypeptide chains( 2

alpha chain and 2 beta chain)

• Collagen – most abundant of all proteins in mammals;

fibrous ; major constituent of skin, tendons, bones, blood

vessels, and other connective tissues

METHODS: ANALYSIS

1. Fractionation

2. Identification

3. Quantitation

Salt Fractionation

Electrophoresis

Immunochemical

SALT FRACTIONATION

• Done by using precipitation reactions

• Globulins are separated from albumins by salting out

• Sodium salts are used to precipitate globulins.

• Not used since direct methods are available.

IMMUNOCHEMICAL METHODS

• Specific proteins are identified with antigen – antibody reactions

• Modified radial immunodiffusion (RID)

• Immunoelectrophoresis (IEP)

• Immunofixation (IFE)

• Immunoturbidimetry

• Immunonephelometry

PROTEIN ANALYSIS: ELECTROPHORESIS

• Protein molecules an be separated by their positive and negative

charges.

• Between electrodes, a positively charged particle will moves toward

the negative electrode and the negatively charged particle will move

towards the positive electrode.

• The overall positive and negative charge of a protein is determined

by how many of the acidic or basic side chain functional groups in the

protein is ionized.

• By changing the medium, proteins can be separated by their

molecular weight.

CHEMICAL PROPERTIES OF PROTEINS

• 1. Protein Hydrolysis

• 2. Protein Denaturation

a. Heat

b. Mechanical agitation

c. Detergents

d. Organic compounds

e. pH change

f. Inorganic salts

CLASSIFICATION OF PROTEINS :FUNCTIONS

• Catalyze reactions -Enzymes

• Regulatory - Hormones

• Storage proteins - Ferritin

• Transport proteins - Hemoglobin

• Structural proteins - Cytoskeleton

• Protective proteins - Antibodies

• Contractile proteins – Myosin ,Actin

SIGNIFICANCE OF PROTEINS

• All biochemical reactions are catalyzed by enzymes, which contain protein.

• The structure of cells and the extracellular matrix that surrounds all cells is

largely made of the protein group collagens. Collagens are the most

abundant protein in the human body.

• The transport of materials in body fluids depends on proteins such as

transferrin , receptors for hormones .

FUNCTIONS OF PROTEINS

• Energy – tissue nutrition

• Osmotic force – maintenance of water distribution between cells and tissues, interstitial compartments, and the

vascular system of the body

• Acid –base – balance – participation as buffers to maintain pH

• Transport – metabolic substances

• Antibodies – part of immune defense system

• Structure – connective tissue

• Enzymes – catalysts

• Hemostasis – participation in coagulation of blood

ENZYMES

• Catalysts for biochemical reactions

• Reactions begin with the migration of the substrate or

substrates into the active site to form ESC.

• Formation of new bonds , second substrate with atoms in the

enzyme.

• Active sites can provide acidic or basic groups without

change in the pH of the environment.

• Enzyme and product molecules separate from each other.

• Enzymes are mostly water – soluble.

•Many enzymes incorporate co factors.( Can either be a metal ion

or a non protein organic molecules – Zn, Mn, Cu, Cr, etc.)

• Enzymes are globular proteins.

FACTORS : AFFECT ENZYME ACTIVITY

•1. Substrate concentration

•2. Enzyme concentration

•3. Temperature

•4. pH of the medium – pH 5-9

•5. Feedback

TERMS:

• Feedback Control – regulation of an enzyme’ s activity by the product

of a reaction later in a pathway

• Allosteric control – an interaction in which the binding of a regulator at

one site on a protein affects the protein’s ability to bind another

molecule at a different site

• Allosteric enzyme – enzyme whose activity is controlled by the binding

of an activator or inhibitor at a location other than the active site

HYPOPROTEINEMIA

• Occurs in condition where a negative nitrogen balance exist.

• Total protein level less than the reference interval.

• Can be cause by excessive loss by excretion in the urine in renal disease , or

leakage in the GI due to inflammation , excessive bleeding (internal or open

wound)

HYPERPROTEINEMIA

• Occurs when there is an underlying cause e.g. dehydration

• When excess water is lost from the vascular system, the proteins, remain within the

blood vessels.

• Dehydration can be cause:

1. Vomiting

2. Excessive sweating

3. Diabetic acidosis

4. hypoaldosteronism

TOTAL PROTEIN METHODS: PRINCIPLES

• Kjeldahl – Digestion of protein; measurement of nitrogen content

• Refractometry – Measurement of the relative index due to solutes in serum

• Biuret – Formation of violet-colored complex chelate between Cu ions and

peptide bonds

• Dye Binding – Protein binds to dye and causes a spectral shift in the

absorbance maximum of the dye

ENZYME REGULATION

• Reversible Noncompetitive Inhibition – an inhibitor binds to an

enzyme elsewhere than at the active site ( reduce activity)

• Reversible Competitive Inhibition- an inhibitor competes with a

substrate for binding to the enzyme active site

• Irreversible Inhibition – enzyme deactivation in which an inhibitor

forms covalent bonds to the active site , permanently blocking it.

CLASSIFICATION:FUNCTIONS

• 1.Hydrolases

• 2.Oxidoreductases

• 3. Lyases

• 4. Ligases

• 5. Isomerases

• 6. Transferases

CHEMICAL MESSENGERS

• HORMONES – controls our vital function; chemical messengers

secreted by cells of the endocrine system

• Via bloodstream

• Via neurotransmitters released by nerve cells

• Chemical receptors –molecule or portion of a molecule which connects

to initiate a response in a target cell.

• Neurotransmitter – a chemical messenger that travels between a

neuron and a neighboring neuron or other target cells to transmit a

nerve impulse

AMINO ACID DERIVATIVES

• Epinephrine

• Norepinephrine

• Thyroxine

• Epinephrine

SOURCE: adrenal medulla , thyroid gland

TARGET: Most cells

MECHANISMS OF ENZYME CONTROL

• 1. Feedback - reactions with reactants, by allosteric control ( bonding –

alter shape- efficiency)

• 2. Inhibition – REVERSIBLE – occur away from the active site(Non

Competitive); COMPETITIVE (at the active site)

• iRREVERSIBLE – covalent bonding is involved

• 3. Production of inactive enzymes – must be activated by cleaving the

portion of the molecule

• 4. Covalent modification of the enzyme by addition or removal of a

phosphoryl group

• 5. Genetic control – regulation of enzyme activity by control of the synthesis

of enzymes

ENZYME INHIBITORS AS DRUGS

• Chemical structures of substrates and the active sites are

known, drug designers can create a molecule that is similar

to the substrate.

• ACE Inhibitors – Angiotensin Converting Enzyme

• Angiotensin II - potent pressor

- elevates blood pressure; contracts

blood vessels

Angiotensin I – inactive precursor of Angiotensin II; 2

amino acids must be cut off to be active ( His and Leu)

ENZYME INHIBITORS: AIDS

•AZT – azidothymidine; Zidovudine

•AIDS fighting drugs

• AZT is accepted by HIV enzyme, it prevents the virus

from producing duplicate copies of itself.

• Ritonovir

• Protease inhibitor

• Dramatic decrease in the virus population

• Success includes a cocktail of drugs with AZT

• Expensive – Cocktail of drugs (20 pills/day)