Xanthoproteic Test Xanthoproteic test is for the aromatic amino acids, tyrosine and tryptophan. The third aromatic amino acid, phenylalanine dose not respond this test.
Principle • On addition of concentrated nitric acid, the white precipitate is formed due to denaturation of proteins. The benzene ring (phenyl groups) present in tyrosine and tryptophan undergoes nitration on heating with nitric acid. The nitro phenyl group imparts yellow colour to the solution. • On addition of the alkali (NaOH), the nitro phenyl group ionizes imparting orange colour to the solution. • Nitration of phenylalanine does not take place under these conditions, therefore it does not respond to this test, although it also contains benzene ring.
Reagents • Concentrated nitric acid. • Sodium hydroxide, (40 % solution in water).
Procedure • Take 2 ml of the given solution in the test tube and add 1 ml of concentrated nitric acid. Mix it, white precipitate is formed. • Heat the solution for 1 minute and then cool under tap water. Some of the precipitate redissolves, and the entire solution becomes yellow. • Add 2 ml of 40% sodium hydroxide solution and mix.
Interpretation • Appearance of deep yellow or orange colour will indicate the presence of aromatic amino acids, tyrosine and tryptophan in the protein. • Gelatin which contains no tryptophan and very little tyrosine, gives only a faint colour.