CHEMISTRY OF PROTENS COLOUR REACTION OF PROTEIN - EXPERIMENTS

 

EXPERIMENTS

 

Egg white is the source of protein used for the following experiments.

 

(1) BIURET test:

                To 1 ml of protein solution add an equal volume of 5% NaOH and 2 drops of 1% copper sulphate, mix, purple or violet colour is produced due to the presence of more than 2 peptide linkage. Tripeptide give +ve test.

 

Explanation:

                Cupric ion in alkaline medium forms a violet coloured complex with peptide bond, nitrogens of peptides and proteins. This reactions is so named, since biuret formed by the condensation of 2 molecules of urea when heated to 180⁰C also answers this test. The minimum requirement for a +ve test is the presence of 2 peptides bonds in the molecule. This is a general test for proteins.

 

Note:

a)       Proteoses and peptones give rose red or pink colour, other proteins give violet colour.

b)       Excess copper sulphate will impart its own blue colour to solution and mask the violet or pink colour of the test.

 

2) Ninhydrine test

 

                To 1 ml of protein solution add 10 drops of ninhydrine reagent, boil and cool. A bluish purple colour is produced.

 

                This test is positive for all alpha amino acids. The test is used in the detection and estimation of amino acids in chromatographic analysis. Proline and hydroxy proline give yellow colour due to imino group (- NH group). This is one of the most sensitive test for amino acids.

Ninhydrin reactions with amino acid is as follows:

Alpha amino acid + Ninhydrin à Aldehyde + CO₂ + NH₃ + Hydrindantin

Hydrindantin + NH₃ + Ninhydrin à Blue complex / purple colour complex.

 

3) Xanthoprotein test

 

                To 3ml of the protein solution add 1ml of conc. HNO3 and boil, solution turns yellow, cool under tap water. Add 40% NaOH drop by drop till it turns alkaline to litmus. Deep yellow or orange colour is developed.

 

                This test is positive for aromatic amino acids like phenyl alanine, tyrosine and tryprophan. Nitration of phenyl ring present in these amino acids give yellow colour characteristic of nitro compounds. In alkaline medium these nitro compounds ionize and produce deep yellow or orange colour.

 

4) Millon’s test

 

                To 1 ml of protein solution add 1.0 ml of million’s reagent. A white precipitate is formed, boil A brick red ppt is formed.

 

                This test is indicative of phenolic aminoacids like - tyrosine, which contains hydroxy phenyl group.

 

                Million’s reagent consists of a solution of nitrate and nitrite of mercury in acid solution, which means nitrous  and nitric acid are also present. Phenolic groups of tyrosine combines with mercury from millons reagent to form mercuric – tyrosine complex which nitration develops red colour.

 

5) Hopkins Cole test: (Aldehyde test): Tryptophan

 

                Mix 1ml of protein solution with 2 drops of 1 in 500 formalin solution and 1 drop of 10% mercuric sulphate. Add gently through the sides of the test tube about 1 ml of Conc. H₂SO4. A purple colour ring develops at the junction of 2 liquids. This indicates presence of tryptophan.

 

Explanation:

                This reaction is due to the presence of Indole ring amino acid tryptophan in the protein molecule. Several aldehdes react with oxidised product of the indole nucleus of tryptophan to give purple coloured complexes (Sulphuric acid with mercuric sulphate is used as oxidizing in this reaction).

 

6) Sakaguchi test:

                To 3 ml of the protein solution add 2 drops of 40% NaOH and 4 drops of Molisch’s reagent mix. Add 2 ml of fresh bromine water. A bright red colour is obtained.

                Substances containing “Ganaidin group” give this colour. Arginine is the only aminoacid in proteins that contains this group. Hence this test is specific for arginine in protein.

                Free arginine or arginyl residues in proteins react with a-naphthol and alkaline hypobronite, to give a bright red coloured complex.

 

7) Sulphur test:

 

                To 1 ml of the protein solution add 1 ml of 40% NaOH and boil for 2 minutes. Cool and add 2, to 3 drops of lead acetate solution. The formation of black or brown precipitate indicates the presence of sulphur containing aminoacids like cystine and cysteine.

 

                Here the hot sod. hydroxide converts-SH or -S.S group present in cystine or cysteine to sod. Sulphide which gives a black precipitate of lead sulphidead acetate, Methionine does not answer this test since the sulfur in methionine is in which is not split with alkali.

 

8) Test For Carbohydrate Moiety in Proteins:

 

Perform Molish’s test: Note the formation of violet ring. Proteins containing carbohydrates groups Glycoprotein and eg. albumin which contains bound carbohydrates answer this test.