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Biochemical Tests for Amino Acids

The biochemical tests for amino acids includes the tests conducted with specific features of the side chains allow to discriminate individual amino acids or some groups of amino acids. It refers to both free amino acids and those bound in peptide of protein.

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Biochemical Tests for Amino Acids

1) Reaction with ninhydrin – a common reaction for all amino acids

Amino acids and peptides, which contain a free α-amino group, react with ninhydrin forming a blue-violet product, whereas proline and hydroxyproline, which contain free imino groups, form a yellow product. Ninhydrin reaction the amino acid undergoes decarboxylation and deamination. The released ammonia (NH3) binds to ninhydrin forming a blue-violet product. This test is due to a reaction between α amino acid group of amino acid and ninhydrin (Triketo hydrindene hydrate). Ninhydrin is a potent oxidizing agent and in its present, amino acid go through oxidative deamination liberating ammonia, carbon dioxide corresponding aldehyde and reduce form of ninhydrin, the NH3 form from α  amino group. The released ammonia binds with ninhydrin and its reduced product (hydrindentane) to give a blue substances diketohydrin (Ruhmen’s purple). However in case of iminoacids like proline, a different product having a bright yellow color is formed. Asparazine has free amide group react to give brown color product. This test is also given by peptides and protein.


2) Characteristic reactions for individual amino acids

A) Detection of Aromatic Amino Acids – Xantoprotein Reaction

The aromatic rings of phenylalanine, tyrosine and tryptophan submitted to the action of nitric acid (HNO3) form yellow nitroderivative products (Ortho nitrotyrosine). This process is known as a Xantoproteic reaction.

Amino acids containing an aromatic nucleus (Tyrosine, Tryptophan, Phenyalanine) form yellow nitro derivatives on heating with concentrated HNO3. The salts of these derivatives are orange color. Protein containing this amino acid also gives a positive response to this test, known as Xanthoproteic test.

B) Detection of Tyrosine – Millon Reaction

Tyrosine reacts with millon reagent like other phenols, which is a mixture of mercury nitrates (V) and mercury nitrates (III) in nitric acid solution. The nitrophenols ensuing from the interaction of tyrosine with nitric acid (V) form red colour complexes with mercury. The mixture of free or peptide bound tyrosine heated with millon reagent results formation of red flocky sediment.

C) Detection of Sulphur Amino Acids – Cysteine Reaction

Cysteine and methionine (Sulphur-containing amino acids): are degraded in strongly alkaline medium, releasing the sulphide ions (S2-), which react with lead (II) acetate forming brown-black lead (II) sulphide.

The guanidine group of arginine reacts with α-naphtol oxidized with bromate (I), releasing ammonia (NH3), and red colour complex.

D) Detection of Tryptophan – Adamkiewicz-Hopkins Reaction

The indole ring of tryptophan reacts with glyoxalic acid – in the presence of sulphuric (VI) acid -forming red-violet product. The glyoxalic acid exists as a contaminating component in commercial preparation of concentrated (glacial) acetic acid.

Indole group of typtophan reacts with glyoxalic acid in presence of concentrated H2SO4 to give purple colour dioxalic acid with magnesium powder or sodium amalgam glacial acetic acid which have been exposed to sunlight also contain gyoxalic acid and thus form red-violet color. This test called as Hopkin’s Cole test.

E) Detection of Histidine – Pauly Reaction

The imidazole ring of histidine, in the presence of sodium nitrite, reacts with sulphanilic acid forming yellow product.

F) Detection of Peptide Bonds – Biuret Reaction

Biuret Test is a general test for compounds having peptides bond. Alkaline copper sulphate reacts with compounds containing two or more peptide bonds to give a violet or pinkish colored product. This is due to formation of coordination complex of cupric ions with unshared electron pairs of peptide nitrogen and oxygen of water.