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JIMENEZ, Zarah Pauline P.

| 2DPh

Section: Plant Lab Protocols

Methodology for Amino Acids and Proteins

Identification of proteins
Proteins are present in the living world, irrespective of the size of the organism, since they form the structural and functional basis of cell. Under certain circumstances, it may become necessary to identify the presence of protein. Isolation of any new compound (e.g., antiviral principle) needs to be attacked primarily through identification reactions. Some of the color reactions of proteins which could be used as identification tests are given below: Test BIURET REACTION To 2mL of the test solution add 2mL of 10% NaOH. Mix. Add two drops of 0.1% CuSO4solution NINHYDRIN TEST To 4mL of the solution which should be at neutral pH add 1mL of 0.1% freshly prepared ninhydrin solution. Mix the contents and boil for a couple of minutes. Allow to cool Observation Violet or pink color Remarks Compounds with two or more peptide bonds give a violet color with alkaline copper sulphate solution The ninhydrin test is answered by amino acids and proteins. The formation of a complex called Rheumanns purple due to condensation of two molecules of ninhydrin with one molecule of ammonia from amino acid is responsible for the violet color. The amino group is the reactive group The yellow color is due to the nitro derivatives of the aromatic amino acids present in the protein. The sodium salts of nitro derivatives are orange in color. The indole group of tryptophan reacts with the glyoxylic acid released by the action of conc. H2SO4 on acetic acid to give a purple color. The guanidine group



Violet or purple color


XANTHOPROTEIC REACTION To 5mL of the solution add 1mL of conc. HNO3. Boil the contents. After cooling ad excess 40% NaOH

On adding acid, yellow color will be noticed. When NaOH is added deep orange color will develop



GLYOXYLIC REACTION FOR TRYPTOPHAN (Hopkins-Cole test) Add 2mL of glacial acetic acid to 2mL of the test solution. Then about 2mL of conc. H2SO4carefully down the sides of the test tube. Observe the color change at the junction of the two liquids. SAKAGUCHI REACTION

Violet ring is formed at the junction

Intense red color


To 5mL of the solution cooled on ice add 1mL of 10% NaOH solution and 1mL of 0.02% -Naphthol solution. After few minutes add 10 drops of alkaline hypobromide solution SULPHUR TEST To 2mL of solution add 2mL of 40% NaOH and 10 drops of 2% lead acetate solution. Boil for a minute and cool.

or arginine reacts with -Naphthol to form a bright red colored complex. Black precipitate The sulphur in sulphur containing amino acids of the proteins in presence of NaOH, is changed into Na2S which forms black lead sulphide when reacted with lead acetate. The yellow precipitate is due to the precipitation of protein. Mercury combines with tyrosine of the protein. The red color is due to reaction of the precipitate with the nitrous acid.


MODIFIED MILLIONS TEST a) To 1mL of solution add 1mL of 10%mercuric sulphate in 10% sulphuric acid. Boil gently for half a minute.

Yellow precipitate

b) Cool under a tap water and add a drop of 1% NaNO2 solution and warm gently

A red color develops ification_of_proteins.php