QUALITATIVE ANALYSIS OF PROTEINS

INTRODUCTION
Proteins are complex macromolecules made up of amino acids produced by all living cells. They have
definite size, shape and charge. They are composed of 20 amino acids in varying number and sequences.
Amino acids contain both a carboxylic group (–COOH) and an amino group (–NH2) and a side chain
(–R Group).The sequence of amino acids determines the characteristics of a protein, which is regulated by
genetic code. All proteins contain C, H, O and N (about 16%); some contain S. The presence of N
differentiates proteins from
carbohydrates and lipids. The proteins perform various functions in the form of enzymes, hormones,
antibodies, coagulation factors, contractile elements and maintenance of osmotic pressure

Reagents and material

1. Protein solution
2. Ethanol
3. Nitric acid
4. Biuret reagent
5. Ninhydrin solution
6. Test tubes
7. Water bath
8. Sodium hydroxide

Reactions of amino acids and proteins can be studied under two broad headings:

A. Precipitation Reactions of Proteins

B. Color Reactions of Proteins

Precipitation reactions of proteins;

Solubility of proteins depends on the proportion and distribution of polar hydrophilic groups and non-
polar hydrophobic groups in molecules. Proteins form colloidal solutions of the emulsion type. A
certain amount of water is imbibed by the protein molecule (water of hydration) and is taken up by the
various free COOH, NH2 and other groups. The stability of the colloidal solution depends on this water
of hydration and on the electric charges carried by the molecules. Thus, removal of the water of hydration
by any dehydrating agents (E.g. Neutral salts and alcohol) will convert the emulsoid or lyophilic colloid
to a suspensoid (Lyophobic colloid). Addition of electrolytes will discharge the protein molecule in
suspension and thus precipitate them.

Principle: The protein molecules exist as anions (negatively charged) in alkaline medium. The negatively
charged protein molecule will combine with the positively charged metallic ions like Ag+, Cd+2, Cu+2,
Fe+3, Hg+2, Pb+2, Sb+3, and Zn+2 and get precipitated.

Precipitation by Ethanol
To 1 ml of protein solution, add 2 ml ethanol. Mix and let it stand for 5 minutes.
A white precipitate is formed

Heller’s Test
Principle: Proteins are denatured by nitric acid with the formation of a white precipitate at the junction of
the two layers (This differs from the nitration reaction in “xanthoproteic acid test”)
Procedure: Take 2 ml of conc. Nitric acid in a test tube. Incline the tube and add, by using a pipette,
equal volume of the protein solution so that the latter forms a layer on the surface of the acid.
A white ring appears at or immediately below the junction of the two fluids. The precipitate does not
dissolve on gentle warming.

Color reaction of protein

The functional groups in amino acids and proteins can react with compounds to produce characteristically
colored products. The intensity of the color formed by a particular group varies among proteins in
proportion to the number of reacting functional or free groups present and their accessibility to the
reacting reagents.

Biuret Reaction
Principle: Cupric ions in an alkaline medium form a violet colored complex with peptide bond nitrogen
of peptides and Proteins. This is a test for peptide linkages. Since all proteins contain peptide linkages,
they respond to this test. The violet color is due to the formation of copper coordination complex between
cupric hydroxide and peptide bond. The tests may also be given by substances containing (– CSNH2), (–
C(NH) NH2), and (– CH2NH2) groups. Biuret, which is obtained by heating urea at 180°C, also gives a
positive test as it contains CONH – linkages. In fact, the name of the test is based on this. Minimum
requirement for a positive test is the presence of two peptide bonds.

Procedure: To 2 ml of protein solution add an equal volume of biuret solution and mix. A violet or
purple color is observed.

Ninhydrin Test
Principle: Ninhydrin reacts with a-amino groups of proteins and free amino acids to give a blue or purple
colored complex. This is one of the most sensitive tests for amino acids answered by all proteins,
peptones, peptides, amino acid and ammonia.

a-Amino acid + Ninhydrin Aldehyde + Hydrindantin CO2 + NH3

Hydrindantin + NH3 + Ninhydrin Blue complex

Procedure: To 1 ml of protein solution, add 2 drops of 0.2% ninhydrin solution and boil for 2 minutes. A
blue color indicates the presence of proteins in the solution. This test is given by all solutions which
contain at least one free amino group and one carboxyl group. Hence, this test is also given by amino
acids in addition to proteins and their derivatives.
Note: Amino acid Proline which has an imino group will produce an orange color instead of the purple-
blue color.

Xanthoproteic Reaction
Principle: On heating with conc. HNO3, proteins containing aromatic amino acids form yellow color due
to the nitration of the benzene ring. The nitro compound is freely ionized in alkaline medium and thus
intensifies the color to orange by adding strong alkali; Tyrosine and
Tryptophan are responsible for this color reaction. Nitration of phenylalanine under these conditions
normally does not take place.
Procedure: Add 1 ml of concentrated HNO3 to 2 ml of solution and boil. Cool the solution and then add
2 ml of 40% NaOH solution till it becomes alkaline. The solution which was acidic with nitric acid turns
orange when it becomes alkaline.
Note: Nitric acid will turn the skin yellow due to the reaction with phenyl groups in the skin. This is not a
toxic reaction but the yellow color doesn’t disappear until
the skin cells are replaced.
Aldehyde Test
(Hopkins-Cole-Adamkiewicz Reaction)
Principle: The indole ring of tryptophan combines with aldehydes, e.g formaldehyde in the presence of
conc. Sulfuric acid to form a violet colored compound. Gelatin gives a negative test as it does not contain
tryptophan.
Procedure: To 1 ml of protein solution, add 1 drop of 40% formaldehyde and mix thoroughly. Then add
1 ml concentrated sulfuric acid along the side of the test tube. It forms a separate layer and a deep violet
or purple ring forms at the junction of the two layers.

Millon’s Test:
Principle: Proteins undergo mercuration and nitration in strong acidic medium to
form red colored mercury phenolate.
The test is given by the Tyrosine amino acid which has OH in the benzene ring.
Note: This test is also given by free phenols and phenolic substances such as
salicylic acid.
Procedure: To 5 ml of protein solution, add 3-4 drops of millon’s reagent. The protein gives a precipitate
of mercury protein complex which adheres to the sides of the test tube. Boil the solution for 1 minute and
cool under tap. The precipitate or solution turns into brick red color.