Proteins:

Proteins are polyamides and a molecular weight above 5000 Kilo Daltons (K.D)

Polyamides of molecular weight below 5000 K.D are usually called poly peptides.

Amino acids are the building blocks of proteins which consist of an amino group, a

hydrogen atom and a typical R group bonded to carbon atom .

Protein Elements

All proteins contain nitrogen. This fact distinguishes them from most

carbohydrates and fats. Also proteins contain carbon; oxygen; hydrogen and a

smaller quantity of sulfur; iodine and phosphates .

General Color Reactions of Proteins reaction

Color producing tests such as the Biuret, Ninhydrin, Millon`s, Hopkins-Cole and

unoxidized sulfur tests are used to detect proteins in biological mixtures. Some of

these reactions [Biuret and Ninhydrin ]are general tests that give positive results

with all proteins and amino acids .

Amino acid

1 .Biuret test :

The biuret test depends on upon the reaction of cupric ions [Cu+2] in an alkaline

solution with peptide linkages of the protein to produce a purple color which is

apparently caused by the coordination complex of copper atom and four nitrogen

atoms of two peptides bonds as shown below :

Procedure

To 1ml of diluted protein solution add 1ml of 10% NaOH mix then add 4-5 drops

of 0.5% CuSO4 and shake, violet color will be produced indicating the presence of

peptide bonds .

protein or poly peptide

Note :

Excess CuSO4 should be avoided because Cu(OH)2will be formed which has

blue color interfering with the violet or pink color of the biuret reaction.

Why the reaction is called as biuret test?

Biuret compound is formed when the Urea is heated. If alkaline cupric ions are

added to biuret solution a violet color is produced. This is a characteristic color not

only of biuret but also proteins and peptides which contain a structure similar to a

biuret. This test requires the presence of at least two peptides linkages per molecule

to be positive. The biuret reaction can be specific to poly peptide since proteins are

about the only compound found in nature that have poly peptide character;

therefore, the biuret test is remarkably specific to proteins .

NH3 + H2N-C-N-C-NH2 2H-N-C-N-H

Biuret compound Urea

2 .Ninhydrin Test:

Ninhydrin reacts with α amino acids to yield a characteristic blue violet

products [decarboxylation]. The overall reaction is shown below:

HO H H O H O

RCHO +

Most amino acids give the same color, except proline makes a pale-yellow

product with ninhydrin. This is due to the fact that proline is an imino acid instead

of having traditional ∝

-amino acids structure.

Ninhydrin proline

Procedure :

Ninhydrin amino acid

decarboxylated amino acid

3H2O,-CO2-

blue color

+yellow color

To 1ml of protein solution that should be neutral or slightly acidic pH 5-7 add

2 drops of freshly prepared 0.2% ninhydrin solution. Boil for 1 minute then allow to

cool. A blue color is produced indicating the presence amino acids or any hydrolysis

product .

3 .Millon`s test:

This test is done to find tyrosine which present in most proteins. Millon`s reagent

contains Hg ion which forms a complex red color with tyrosine. If the unknown is

protein solution, a red precipitate will be formed due to the heavy metals (Hg) are

precipitating agents, while if the unknown is tyrosine solution then a red solution

will be formed .

Note :

Excess chloride ions interfere with this test combing with Hg ions present in

Millon`s reagent so this test cannot be used to detect tyrosine or protein in urine

since urine contains a significant amount of chloride ions. Millon`s test will be

Tyrosine

conducted with one compound which is not an amino acids nor a protein. This

compound is salicylic acid which a simply a 2-hydroxy benzoic acid.

Procedure:

To 1ml of unknown solution add 1 drop of Millon`s reagent and heat gently. Red

precipitate or solution indicates the presence of protein or tyrosine.

4 .Glyoxylic Acid Test(Hopkin`s Cole Reagent)

This test is done to detect the presence of tryptophan amino acid, which contain

indole group. Strong acids such as H2SO4 oxidize glyoxyl (CHO—CHO) to

glyoxylic acid then this compound condenses with tryptophan (indole ring)forming

red, purple, violet or yellow color complex .

Note:

1 .glacial acetic acid usually contains some glyoxylic acid as an impurity; therefore,

it is as suitable source glyoxylic acid and glyoxyl .

2 .H2SO4 used should be pure, otherwise color will not develop.

Procedure:

To 1 ml of glacial acetic acid which contains glyoxylic acid add 1ml of unknown

solution, mix then incline the test tube and slowly slide 1 ml of concentrated H2SO4

down its side so that the sulfuric acid forms a distinct bottom of the test tube. A red

or yellow color as ring will appear indicate the presence of tryptophan.

5 .Unoxidized Sulfur Test:

This test is positive only in the presence of amino acid containing sulfhydryl

(SH)or disulfide S-S group.

Methionine is the third amino acid which contain sulfur but not in the form of

sulfhydryl (SH)or disulfide S-S group .

When proteins boiled in strong alkali, the SH and S-S groups are converted

into inorganic sulfide. If we add lead acetate solution black precipitate of lead

sulfide PbS is formed.

NH3

PbS inorganic sulfideHS-CH2-CH-COO

Procedure:

NaOH

100 C ͦ

Pb (CH3COO)2

1.To about 1 ml of unknown solution add 2 ml of 40% NaOH .

2.Boil for (10min) to convert the organically combined sulfur to inorganic form .

3 .Remove from heat and add 10 drop of lead acetate.

4 .Brown or black precipitate of lead sulfide will form indicating the presence of

cysteine and cystine .

Cysteine Cystine Methionine

Five qualitative color test of proteins are summarized in the following table

Specific for materialReagent usedName of test

Poly peptide and

proteins

Alkaline CuSO4 in 10%

sodium hydroxide

Biuret

Amino acidNinhydrin in water

saturated butanol

Ninhydrin

Phenolic hydroxyl

group of tyrosine

Mercuric and mercurous

nitrates in HNO3

Millon

Indole group of

tryptophan

Glyoxylic acid in H2SO4Hopkins-cole

SH and SS groups in

cystine and cysteine

respectively.

Strong NaOH and

acetate

Unoxidized

Physical reaction

1. Solubility of protein

2 .Precipitation of protein

3. Coagulation of protein

1.Solubility of protein

The solubility of amino acids and proteins is largely dependent on the solution

pH. As amino acids have both an “amino” group and a “carboxylic” group, they are

considered as both “base” and “acid”, i.e. they are amphoteric the protein will

soluble in the basic and acidic medium the protein while in neutral medium the

protein is precipitate .

PeptonCaseinGelatinAlbumin

Not solubleNot solubleNot solubleNot solubleCold H2O

Semi solubleSemi solubleSemi solubleSemi solubleHot H2O

SolubleSolubleSolubleSoluble10% NaOH

SolubleSolubleSolubleSoluble10%HCl

2.Precipitation of protein

The charge of protein in the acidic medium will be (+ve ) while in the basic

medium the charge will be (-ve) and the protein is soluble. while in neutral medium

the protein is precipitate .

R-C-H

OH- COO-

CH-COO - - NH2 R

Zwitterion

a . Precipitation by Heavy Metal

In most naturally occurring protein solutions the protein molecules are negatively

charged. Neutralization of this charge bring proteins to the isoelectric point. At this

point, maximum precipitation of proteins take place and the protein particles bear

zero net charge. Each protein has its own isoelectric point, since they may be

precipitating by providing the positively charged molecules from another reagent.

salts of heavy metals like iron, copper, zinc, mercury, silver, etc. are very suitable

for this, however, addition of excess amount of salts of heavy metals solutions to

protein hence these particles may become dissolve again .

The precipitation of protein by salts of heavy metals is also due to at least partly to

formation of insoluble compounds of proteins and the metallic cations for example

NH3+

H+

CH-COOH-+NH3

R

when we add {Pb+2, Hg+2} to casein or albumin, lead caseinate or s a mercury

albumin may form as a precipitate .

H H

R-C-COOAg Ag + + R-C-

COO -

silver proteinate NH2 NH2

at isolectric point

white PPt

Procedure

To 1ml (alb. or peptone) add 3 drops of CuSO4 or FeCL3 or Pb(CH3COO)2 or

AgNO3

b. Precipitation by alkaloid reagents

The addition of an acid to a protein solution causes the protein particles to acquire

a positive charge. By the cautious addition of an alkaloidal reagent (which provides

complex negatively charged ions) to the acidified protein solution, the protein can

be predicated at the isolelectric point. An excess of the reagent may the precipitate

to disappear by providing the protein particles with an effective negative charge.

Alkaloidal reagents include:(sulphosalicylic, metaphosphoric, tannic,

phosophotungtic and trichloro acetic acids .

Procedure

white ppt 1ml of protein + 1ml of phosophotungtic acid

c. Precipitation by neutral salts

Protein molecules contain both hydrophilic and hydrophobic amino acids. In

aqueous medium, hydrophobic amino acids form protected areas while hydrophilic

amino acids form hydrogen bonds with surrounding water molecules (solvation

layer). When proteins are present in salt solutions (e.g. ammonium sulfate), some of

the water molecules in the solvation layer are attracted by salt ions. When salt

concentration gradually increases, the number of water molecules in the solvation

layer gradually decreases until protein molecules coagulate forming a precipitate;

this is known as “salting out”. As different proteins have different compositions of

amino acids, different proteins precipitate at different concentrations of salt solution.

1 .Half saturation (reversible ppt)

1ml pr.+1ml ammonium sulfate solution

white ppt or turbid

No ppt albumin

pptglobin

The reason for the precipitation of globulin and albumin at different ammonium

sulfate concentration could be that the solvation layer around globulin is looser and

thinner than that around albumin. Therefore, globulin needs only half-saturated

ammonium sulfate to lose its solvation layer while albumin loses its solvation layer

in a fully saturated ammonium sulfate solution.

2 .Complete saturation

white ppt or turbid 1ml protein + solid ammonium sulfate

3 .Denaturation (coagulation):(irreversible precipitation )

Heat disrupts hydrogen bonds of secondary and tertiary protein structure

while the primary structure remains unaffected. The protein increases in size due to

denaturation and coagulation occur.

Denaturation : is a process in which the biological activity of protein is lost there are many factors such as

1 .heating

2 .mixing

3.x-ray

4 .ultrasonic vibration

Procedure

Add 3 ml of albumin or globulin then 2-3 drops of acetic acid

heating

precipitation ve+ albumin, globulin

casein, pep., gel . - ve

Unknown of protein :

Biuret test

- ve amino acid ve +

or non proteins substanceprotein

Ninhydrin test

non protein ve - ve aminoacid + +ve turbid or white ppt alb.or glob

Sulfur test (cystein and cystine ) -

Hopkin`s cole test(tryptophan)-

clear solution (-ve)

pepton, casien, gelatin

Denaturation test