Experiment 172

Objective:  Test for Aromatic Rings in Proteins

Introduction:

Using 65% nitric acid, the aromatic rings in amino acids such as tyrosine and tryptophan undergo nitration, forming nitro derivatives that show an intensely yellow color. Since most proteins contain aromatic amino acids, this reaction is used as a general test for the presence of proteins.

The xanthoproteic reaction explains why nitric acid causes yellow stains on the skin: epidermal cells contain aromatic proteins that react with nitric acid. Additionally, peptides and proteins react with Cu²⁺ in an alkaline environment to form a blue-violet colored chelate complex, known as the biuret test, which has an absorbance maximum at 540 nm.    

The intensity of the color in the biuret test is proportional to the number of peptide bonds, and it requires at least three peptide bonds to occur.

Chemicals:

AlanineTyrosineEgg-whiteHNO₃ (65%)

Glassware:

3 conical measures, graduated (250 mL)3 beakers (250 mL)Beaker (50 mL)2 snap-cap vials (10 mL)3 glass stirring rods

Experimental Procedure:

Set up three conical measures as described below, and mix the solutions with a glass stirring rod.

Observations:

Conical Measure 1: Solution remains colorless.Conical Measure 2: A yellow color is clearly visible.Conical Measure 3: An intensely yellow-colored precipitate form

Questions:

1.    What change occurred in the test? The yellow color observed in conical measures 2 and 3 indicates nitration of aromatic amino acids like tyrosine and tryptophan.

2.    Which proteins are used in the reaction? Tyrosine and egg-white (which contains aromatic proteins) are used in the reaction.

3.    What amino acids are present in the test? The amino acids tyrosine and tryptophan, which contain aromatic rings, are the focus of this test.

4.    Why is 65% HNO₃ used? 65% nitric acid is used to nitrate the aromatic rings in the amino acids, leading to the formation of yellow-colored nitro derivatives, which is characteristic of the xanthoproteic reaction.