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Color Reactions to Proteins and Individual Amino Acids

In this color reactions to proteins and individual amino acids post we have briefly explained about color reactions of amino acids such as ninhydrin, biuret reaction, xanthoproteic test, etc.

Color Reactions to Proteins and Individual Amino Acids

Proteins are made up of peptide bonds that connect amino acid residues. Amino acid reacts with a range of chemicals to create coloured products due to its polypeptide structure and varied amino acid residues. These color reactions of amino acids tests, also known as amino acid colour reactions, are useful for detecting and quantifying amino acids, as well as their constituent amino acids, in body fluids and other biological materials.

Ninhydrin Reaction

All amino acids when heated with ninhydrin can form complexes; pink, purple or blue in color. The color reactions of amino acids is called Ruhemann’s purple. Proline and hydroxy proline will give yellow color with ninhydrin. Amino acids with amide group (glutamine, asparagine) produce a brown color.

The ninhydrin reaction may be adopted for qualitative as well as quantitative estimation of amino acids. It is often used for detection of amino acids in chromatography. Proteins do not give a true color reaction; but N-terminal end amino group of protein will also react with ninhydrin, to produce a blue color.

Biuret Reaction

Cupric ions in an alkaline medium form a violet color with peptide bond nitrogen (Schiff, 1896). This needs a minimum of two peptide bonds, and so individual amino acids and di-peptides will not answer this test. Color reactions of amino acids can be used for quantitative estimation also. 

The name is derived from the compound biuret (NH2—CO—NH—CO—NH2), a condensation product of two urea molecules, which also gives a positive color test. Magnesium and ammonium sulphates interfere with this reaction.

Xanthoproteic test

The ring systems in phenyl alanine, tyrosine and tryptophan undergo nitration on treatment with concentrated nitric acid when heated (Salkowski, 1888). The end product is yellow in color which is intensified in strong alkaline medium. This color reactions of amino acids causes the yellow stain in skin by nitric acid.

Millon’s Test

The phenol group of phenylalanine and tyrosine containing proteins, when heated with mercuric sulphate in sulphuric acid and sodium nitrite (or, mercurous and mercuric nitrates in nitric acid) form red coloured mercury phenolate (Millon, 1849). Chloride interferes with this color reactions of amino acids and so it is not suitable to test for tyrosine in urine samples. Both xanthoproteic and Millon’s tests are negative for tapioca (casava) which is deficient in phenylalanine and tyrosine.

Aldehyde Tests

In the Hopkins-Cole test, tryptophan containing protein is mixed with glyoxylic  acid, and the mixture is layered over concentrated sulphuric acid. A violet ring at the interface of liquids shows the presence of the indole ring. Formaldehyde and mercuric sulphate is used similarly in Acree-Rosenheim reaction to get a violet color reactions of amino acids. Para-dimethyl-amino-benzaldehyde and strong hydrochloric acid give dark blue color (Ehrlich’s reaction). Gelatin with limited tryptophan content will not answer these tests.

Sakaguchi’s Test

Free arginine or arginyl residues in proteins react with alpha-naphthol and alkaline hypobromite to give bright red color. This color reactions of amino acids is due to the guanidinium group.

Sulphur Test

When cysteine or cysteine containing proteins are boiled with strong alkali, organic sulphur splits and forms sodium sulphide, which on addition of lead acetate produces lead sulphide as a black precipitate color reactions of amino acids. Methionine does not answer this test because sulphur in methionine is in the thio-ether linkage which is difficult to break. Albumin and keratin will answer sulphur test positively; but casein will give a negative test color reactions of amino acids.

Nitroprusside Test

Proteins with free sulfhydryl groups give a reddish color with sodium nitroprusside, in ammoniacal solution color reactions of amino acids. Many proteins give a negative, reaction in the native state, but when denatured, reaction will be positive, showing the emergence of free SH groups.

Pauly’s Test

Diazo benzene sulfonic acid reacts with imidazole group of Histidine to form a cherry-red coloured diazotised product under alkaline conditions color reactions of amino acids. The same reagent will give an orange red coloured product with phenol group of Tyrosine.

Further Readings


Color Reactions to Proteins and Individual Amino Acids