This test used for detecting the presence of tryptophan in proteins.

A violet zone in the junction point of two layers is a positive result.

A similar result is obtained by the Hopkins-Cole reaction which is frequently used to detect Trp in a protein solution.

Observe the formation of purple color at the interface between the two layers.

Central Department of Microbiology (Tribhuvan University(TU), Nepal).

Central Department of Biotechnology (Tribhuvan University (TU), Nepal).

Each amino acid consists of: Central carbon atoms An amino acid Carboxyl group Side chain Different side chain result in various amino acid 1.

H, the carboxyl group is deprotonated but the amino group is protonated.

The net charge is zero; such ions are called Zwitterions At alkaline p.

H, the amino group is neutral NH 2 and the amino acid is in the anionic form.

This reaction provides an extremely sensitive test for amino acids.

While most ninhydrin tests result in a purple color, the proline reaction is more yellow due to substitution of the alpha amino group that ninhydrin reacts with carbon rings.

The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction.

The working range for the biuret assay is 5 -160 mgm.

L. What is chelation chelation involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand a single central atom.

Consequently, any protein containing tyrosine will give a positive test of a pink to dark-red color.

The reagent used in this test is called Millons reagent and it contains mixture of Hg, Hg 2, HNO 3 and HNO 2.

BWB 5 min A white ppt is formed with albumin and casein (but not gelatin); the ppt gradually turns into brick red.

These aromatic groups can undergo reactions One such reaction is the nitration of a benzene ring with nitric acid.

The amino acids that have activated benzene ring can readily undergo nitration.

In the presence of activated benzene ring, forms yellow product.

Apply this test to tyrosine, tryptophan, phenylalanine and glutamic acid.

OH (why) Observe the color of the nitro derivative of aromatic nucleus.

Once the tryptophan is free, it reacts with glyoxylic acid to form violet product.

H 2 S 04 acid flow slowly down the side of the test tube, thus forming a reddish - violet ring at the interface of the two layers.

This reaction is due to partial conversion of the organic sulfur to inorganic sulfide, which can detected by precipitating it to lead sulfide, using lead acetate solution.

S. (protein) 2 Na.

OH---- Na 2 S (CH 3 COO)2 pb ------- Pb.

Add 5 drops of 10 lead acetate solution.

Stopper the tubes and shake them.

Remove the stoppers and heat in a boiling water bath for 5 minutes.

Hopkins Cole Reagent