COLOUR REACTIONS OF PROTEINS

BIURET TEST XANTHOPROTEIC TEST COLE’S MERCURIC NITRITE TEST HOPKINS COLE (ALDEHYDE) TEST SAKAGUCHI’S TEST SULPHUR TEST MOLISCH TESTS

BIURET TEST (GROUP TEST FOR PROTEINS) PRINCIPLE: proteins + biuret reagent purple coloured complex which is due to coordination complex between cupric ions and nitrogen atoms of the peptide bond (CO-NH). all proteins and peptides possessing at least two peptide bonds answer biuret test. ProcedureObservationInference To 2ml of albumin solution add 2ml of 5% sodium hydroxide solution and mixViolet colour appearsAlbumin is a protein. well. Then add 3 to 4 drops of 1% copper sulphate.

XANTHOPROTEIC TEST ( For aromatic amino acid) PRINCIPLE: Nitration of benzene ring gives yellow color which turns to orange color in alkaline medium due to formation of sodium salt of nitrobenzene. i)To 3 ml of egg albumin add 1 ml of conc. Nitric acid. Boil for a minute and then cool it under tap water. ii)Add 5 drops of 40% sodium hydroxide. Yellow precipitate is formed Yellow precipitate turns orange. presence of aromatic amino acid in egg albumin.

COLE’S MERCURIC NITRITE TEST (MODIFIED MILLON’S REACTION ) PRINCIPLE: Mercury forms complex with phenolic hydroxyl group of tyrosine to form mercuric phenolate, which in the presence of sodium hydroxide form sodium salt of mercuric phenolate. i) To 1ml of albumin add 1mlsyellow precipitate is formed of 10% mercuric sulphate in 10% sulphuric acid. Boil for 1 Presence of phenolic group of minute. Cool the test tubes. tyrosine in albumin ii) Add 5 drops of 1% sodiumYellow precipitate turns red nitrite.

HOPKINS – COLE’S TEST (ALDEHYDE TEST) PRINCIPLE: The indole group of tryptophan forms purple colour with formaldehyde in the presence of mercuric sulphate in acidic medium. To 2ml of albumin, 2 drops of 1/500 formalin and 1 drop of 10% mercuric sulphate in 10% sulphuric acid is added and mixed well. Then 2ml of concentrated sulphuric acid is added along the sides of the test tube. purple ring is formed at the junction of two layers Presence of tryptophan in albumin

SAKAGUCHI’S TEST PRINCIPLE: Guanido group of arginine gives red colour with the reagents. Sakaguchi reagent consists of 1-naphthol and a drop of sodium hypobromite To 3ml of albumin, add 5 drops of 40% sodium hydroxide, 2-3 drops of 1% alpha naphthol.Caramine red colour isPresence of arginine in Mix well and add 10 drops offormed.albumin. freshly prepared bromine water.

SULPHUR TEST PRINCIPLE Sulphur group combines with sodium hydroxide to form sodium sulphide which reacts with lead acetate to form black lead sulphide precipitate. a) To 2ml of albumin add 2mlWhite precipitate is formed of 40% sodium hydroxide, mix well, and boil for a minute. Presence of sulphur containing b) Then add 2-3 drops of leadBlack precipitate is formedamino acid in albumin. acetate.

MOLISCH’S TEST PRINCIPLE: Carbohydrates in the glycoprotein undergo dehydration in the presence of conc. Sulphuric acid to form furfural which gives purple colour ring with alpha naphthol. To 2ml of albumin solution, 3 drops of Molisch’s reagent is added and mixed. The test tube is inclined and 2 ml of conc. Sulphuric acid is added along the sides of the tube. Violet colour ring is formed at the junction of two liquids. Egg albumin is a glycoprotein.

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