Color reactions of Proteins : Biochemistry series

The Biuret test detects peptide bonds in proteins. Add 1 ml of protein solution to a test tube, followed by 1 ml of 5% sodium hydroxide and 2-3 drops of 1% copper sulfate solution. Initially, a blue precipitate forms, which turns violet upon mixing, indicating a positive result. This reaction requires at least two peptide bonds; individual amino acids and dipeptides will not react. Excess copper sulfate should be avoided.

The Ninhydrin test detects alpha amino acids. Add 10 drops of ninhydrin solution to 1 ml of protein solution and heat until it boils strongly. The formation of a purple color indicates a positive result, due to the formation of Ruhemann's purple. This reaction is also used in forensic medicine for fingerprint detection.

The Xanthoproteic test detects aromatic amino acids like tyrosine and tryptophan. Add 1 ml of concentrated nitric acid to 2 ml of protein solution, mix thoroughly, and boil for about one minute. A yellow color forms due to nitro derivatives. Divide the solution and add 1 ml of 40% sodium hydroxide to one half until it turns alkaline (turns red litmus blue). The yellow color will change to orange, indicating the presence of aromatic amino acids. Phenylalanine typically does not react under these conditions.

Millon's test, or the Cole test, detects tyrosine. Add 1 ml of Millon's reagent (mercuric nitrate in nitric acid) to 1 ml of protein solution. Gently boil for 30 seconds, then add two drops of 1% sodium nitrite. The color formed is due to the formation of nitrated mercuric phenolate ion of tyrosine.

The Sakaguchi test detects the guanidine group, specific to the amino acid arginine. To 3 ml of protein solution, add two drops of 40% sodium hydroxide, then four drops of alpha naphthol solution, and finally 10 drops of bromine water. The formation of a red precipitate indicates a positive test. Alternatively, sodium hypochlorite can be used instead of bromine water.

The Sulfur test detects sulfur-containing amino acids مثل cysteine. Add an equal volume of 40% sodium hydroxide to 1 ml of protein solution and boil for at least three minutes. After cooling, add 2-3 drops of lead acetate solution. A brown to black precipitate indicates the presence of sulfur. Methionine does not give a positive result as its sulfur linkage is not easily broken by sodium hydroxide.

Pauli's test detects the imidazolic group and phenolic hydroxyl group, present in histidine and tyrosine, respectively. Mix 0.5 ml of sulfanilic acid with 0.5 ml of 0.5% sodium nitrite. After one minute, add 1 ml of protein solution, then 2 ml of 10% sodium carbonate to make the solution alkaline. Observe the formation of a cherry-red color.

The Aldehyde test, also known as the Hopkins-Cole test, detects the indole nucleus present in tryptophan. To 1 ml of protein solution, add a drop of formaldehyde, then a drop of mercuric sulfate solution. While holding the test tube slanted, add 1 ml of concentrated sulfuric acid. A violet-colored ring at the junction of the two liquids indicates a positive result. This occurs due to the oxidation of the indole group in tryptophan reacting with the aldehyde.