Color Test for Proteins
Proteins exhibit characteristic colour reactions when treated with certain reagents. Some of these reactions depend upon the presence of particular amino acids in the protein. Others are due to the peptide linkages between the amino acids. It may thus be necessary to try several tests before deciding whether a subtance is a protein or not. Summary is given in table below.
Xanthoproteic Test
When a protein is treated with concentrated nitric acid, a yellow colour is produced. The colour is turned to orange if the solution is made basic. Any one who has spilled nitric acid on his hands will recall the yellow colour produced by the reaction of the nitric acid with the protein of the skin. This test is given by a protein that consists of amino acids containing a benzene ring, such as tyrosine or phenylalanine.
Biuret Test
When a protein is treated with a small amount of sodium hydroxide solution and a dilute solution of cupric sulphate is added drop by drop, a violet colour is produced. This test is given by all substances that contain two or more peptide linkages, that is, such substances as proteins or polypeptides. The colour is due to the formation of a coordination complex between Cu2+ and >C=0 and -NH- groups of the peptide linkages. The biuret test is negative for amino acids, which do not contain a peptide linkage, or for dipeptides which contain only one peptide linkage.
Millon's Test
Millon's reagent consists of mercury dissolved in nitric acid (forming, a mixture of mercuric and mercurous nitrates). When Million's reagent is added to a protein, a white precipitate forms. This white precipitate on heating turns to a brick-red colour This test is given by those proteins which yield tyrosine on hydrolysis.
Hopkins-Cole Test
To a mixture of protein and a glyoxalic acid solution, concen- treated sulphuric acid is added carefully down the sides of the test tube to form a layer. If tryptophan is present in the protein, a violet ring will appear between the two layers.
Sulphur Test
When a sulphur-containing protein is heated with a mixture of sodium hydroxide and lead acetate solutions, a black precipitate of lead sulphide is formed.
Ninhydrin Test
When proteins are boiled with a dilute aqueous solution of ninhydrin, a purple colour is produced. This test is given by α-amino acids, proteins and dipeptides.
| Colour Reactions of Proteins/Amino Acids | |
|---|---|
| Reaction | Specific Group or Amino Acid |
| Biuret reaction | Two peptide linkages |
| Ninhydrin reaction | α-Amino acids |
| Xanthoproteic | Benzene ring of aromatic reaction amino acids (Phe, Tyr, Trp) |
| Milllons reaction | Phenolic group (Tyr) |
| Hopkins-Cole reaction | Indole ring (Trp) |
| Sakaguchi reaction | Guanidino group (Arg) |
| Nitroprusside reaction | Sulfhydryl groups (Cys) |
| Sulfur test | Sulfhydryl groups (Cys) |
| Pauly's test | Imidazole ring (His) |
| Folin–Coicalteau's test | Phenolic groups (Tyr) |
Source: Advanced Organic Chemistry By B.S.Bahl and Arun Bahl
