NINHYDRIN TEST

BACKGROUND Ninhydrin test is one of the most sensitive test for amino acids and is given positive by all the free amino acids, proteins, peptones as well as ammonia and other primary amines. It is used for detection of all amino acids expect proline and hydroxyproline. Because they don’t have free amino group.

OBJECTIVE To detect alpha amino acids in given solution.

PRINCIPLE Ninhydrin is a powerful oxidizing agent and causes oxidative decarboxylation of α-amino acids producing CO2 and ammonia. The reaction of ninhydrin with amino acids is as follows α-amino acids + ninhydrin → aldehyde + CO2 + NH3 + Hydindantin Hydindantin + NH3 + ninhydrin → blue complex

Ninhydrin is reduced to hydindantin during reaction with the α-amino group. The amino acid in turn is converted to an aldehyde, ammonia and CO2. hydindantin and ammonia interact with another molecule of ninhydrin to form a purple coloured product.

REAGENTS Ninhydrin solution, 0.1%

PROCEDURE Take 1ml of given solution and add 2 drops of 0.1% ninhydrin solution. Mix and boil for 1-2 minutes and allow it to cool.

INTERPRETATION A blue color develops if test is positive. Proline and hydroxyproline give a yellow color.

PRECAUTIONS While performing the test with casein or alpha keratin which are dissloved in alkaline solutions, add few drops of 1% acetic acid to neutralize and then perform ninhydrin test.

SULPHUR TEST

OBJECTIVE This test is used for sulphur containing amino acid cysteine. Cysteine is a neutral amino acid.

PRINCIPLE When cysteine or cystine or proteins containing these amino acids are boiled with strong alkali, e.g., sodium hyroxide, the sulphur present in the protein is liberated as sodium sulphide. This reacts with lead acetate to frm a brown or black precipitate of lead sulphide. R-SH + 2NaOH → R-OH + Na2S + H2O Na2S + (CH3COO)2Pb → PbS ↓ 2CH3COONa

Cysteine and cystine respond to this test but the sulphur present in methionine is not released by the above treatment, therefore, methionine does not answer this test. Casein and gelatin, which contain methionine but practically negligible amounts of cysteine give a negative test.

REAGENTS Sodium hydroxide, 40% solution in water Lead acetate, 2% soultion in water

PROCEDURE Take 2ml of the given solution in a test tube and add 2ml of 40 % sodium hyroxide solution and boil for two minutes. Add 1ml of lead acetate solution and then warm slightly.

INTERPRETATION A brown or black precipitate forms if cysteine or protein containing cysteine is present in the given solution.