BIURET TEST

BACKGROUND: Colour reactions are due to a reaction between one or more constituents radicals or group of radicals or group of complex protein molecules and the chemical reagent used in any given test. Most of the color reactions are not used to determine the protein in urine because urine itself is colored and secondly its inorganic constituents is colored and secondly its inorganic constituents e.g. sulphates interfere with the color reactions

OBJECTIVE To detect the presence of proteins in the given soultion. It is a general test for proteins.

PRINCIPLE Biuret test is a general test for proteins. It gives color reaction due to presence of peptide linkage in polypeptide or protein since all protein contain peptide linkages. Compounds containing two or more peptide bonds give a characteristic purple colour when treated with dilute copper sulphate in an alkaline solution. The name of the test comes from the compound biuret, which is the simplest compound that gives a typical positive reaction.

The structure of biuret shows clearlythat it is formed from two molecules of urea, when heated at 180 C. the exact reason of the color is not known. According to one common view, the color is due to the coordination complex of the copper atom with four nitrogen atoms of a protein.

REAGENTS SODIUM HYDROXIDE: 10 % SOLUTION IN WATER COPPER SULPHATE:0.5% SOULTION IN WATER

PROCEDURE Take two test tubes and label them “T” (for test) and “B” (for blank). Take 2ml of the protein solution in “T” test tube and 2ml of the distilled water in “B” test tube. Add 2ml of sodium hydroxide in both test tubed. Mix and add copper sulphate solution drop by drop, mix after each addition by inversion. Usually 4-6 drops of copper sulphate solution are sufficient to obtain good results.

interpretation A purpulish violet or pinkish violet color indicates the presence of protein in “T” test tube. If the color becomes blue, it indicates negative test. Peptone gives pink color, and may require the addition of a larger amount of copper sulphate solution than usual. Gelatin may give a very faint color and, therefore, control is required to avoid confusion.

PRECAUTIONS Excess of copper sulphate should be avoided, otherwise, the blue color of cupric hydroxide will mask the violet color. Magnesium sulphate and ammonium sulphate interfere with this test. Therefore, the test should not be carried out with solutions containing these salts.

LIMITATIONS The test may also be given by substances containing a peptide bond whether it is in protein or in non-protein compound, such as biuret and oxamide.

XANTHOPROTEIC TEST

OBJECTIVE Xanthoproteic test is for the detection of aromartic amino acids, tyrosine and tryptophan. The third aromatic amino acid, phenylalanine does 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 group) present in tyrosine and tryptophan udergoes nitration on heating with nitric acid. The nitrophenyl group imparts yellow colour due to the solution. On addition of the ammonia (NH3), the nitrophenyl group ionizes imparting orange color to the solution. Nitration of phenylalanine does not take part under these conditions, therefore it does not respond to this test, although it also contains benzene ring.

REAGENTS Concentrated nitric acid Sodiun hydroxide, (40% soultion in water)

PROCEDURE Take 2ml of the given soultion in a test tube and add 1ml 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 few drops of ammonia and mix.

INTERPRETATION Appearance of deep yellow or orange color 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 color.