Organic Chemistry

Module 6. Proteins

Lesson 19
QUALITATIVE TEST FOR PROTEINS

19.1 Introduction

As the name suggest amino acids are organic compounds that contain amino and carboxyl groups. The R- in the above formula stands for different chemical groups (may be aliphatic, aromatic or heterocycylic) and this determines the characteristics of the amino acids.The color tests have frequently been used for qualitative detection of amino acids. Not all amino acids contain the same reactive groups. For this reason the various color tests yield reactions varying in intensity and type of color according to the nature of groups contained in the particular amino acid under examination.

19.2 The Detection of Proteins

19.2.1 Millon’s reaction

19.2.1.1 Principle

The reaction is due to the presence of the hydroxyphenyl group, C₆H₅OH in the amino acid molecule; and any phenolic compound which is unsubstituted in the 3,5 positions such as tyrosine, phenol and thymol will give the reaction. Solutions of nitric acid containing mercuric nitrate reacts with phenols, producing red colors or yellow precipitates which react with nitric acid to form red solution. The reaction probably depends on the formation of a nitro compound; which then reacts with phenol.

19.2.1.2 Materials

1. Millon’s reagent

19.2.1.3 Method

Add 3 to 4 drops of Millon’s reagent to 5 ml of test solution. Mix and bring the mixture gradually to a boiling point by heating over a small flame. Development of red color is due to the presence of protein. Excess of reagent should however be avoided since it may produce a yellow color which is not a positive reaction.

19.2.2 Millon-Nasse reaction

19.2.2.1 Materials

1. Millon-Nasse reagent
2. 1% NaNO₂

19.2.2.2 Method

Add 1 ml of Millon-Nasse reagent to 5 ml of test solution. Place the tube in a boiling water bath for 10 mins. and cool the contents in water bath for 5 to 10 mins. and add 1 ml of 1% NaNO₂. A deep red color indicates tyrosine or other 3,5 unsubstitued phenol.

19.2.3 Xanthoproteic reaction

19.2.3.1 Principle

This reaction is due to the presence in the amino acid molecule of the phenyl group –C₆H₅, with which the nitric acid forms certain nitro modifications. The particular amino acids which are of especial importance in this connection are those of tyrosine and tryptophan. Phenylalanine does not respond to this test as it is ordinarily preformed.

19.2.3.2 Materials

1. Conc. HNO₃
2. Ammonium hydroxide
3. Sodium hydroxide

19.2.3.3 Method

Add 1 ml of conc. Nitric acid to 2 to 3 ml of test solution in a test tube. A white precipitate forms, which upon heating turns yellow and finally dissolves, imparting to the solution a yellow color, cool the solution and carefully add ammonium hydroxide or sodium hydroxide in excess. Note that the yellow color deepens into an orange.

19.2.4 Hopkins-Cole reaction

19.2.4.1 Principle

The formation in this test color is due to the presence of indoyl group. Gelatin does not respond to this test due to lack of amino acid tryptophan. Violet to blue colors develop when a mixture of protein and an aldehyde is layered over conc. sulphuric acid. A number of tests based on this principle have been suggested; all depends on the presence of the indoly group of tryptophan which reacts as follows (using glyoxylic acid as an example of an aldehyde).

This is called Hopkin-Cole test- A similar test was at one time recommended for detection of formaldehyde that had been as a preservative to milk, the formaldehyde reacting with indolyl groups of milk proteins to give a color.

19.2.4.2 Materials

1. Hopkin-Cole reagent
2. Conc. H₂SO₄

19.2.4.3 Method

Place 2 to 3 ml of test solution and an equal volume of Hopkins-Cole reagent in a test tube and mix thoroughly. Incline the tube and permit 5 to 6 ml of conc. sulphuric acid to flow slowly down the side of the tube, thus forming a sharp layer of acid beneath the amino acid solution. When stratified in this manner a reddish-violet color forms at the zone of contact of the two fluids. If the color does not appear after starting for a few minutes, the tube may be rocked gently to cause a slight mixing of the liquids are mixed by gentle stirring the precipitate of protein dissolves and the violet color spread throughout the solution.

19.2.5 Biuret test

19.2.5.1 Principle

The Biuret test is given by those substances whose molecules contain two cabamyl (-CONH₂) groups joined either directly or through a single atom of nitrogen or carbon. Similar substances which contain- CSNH₂,

-C(NH)NH₂, or – CH₂NH₂ in place of the –CONH₂ group also respond to the test. It follows from this fact that substance which are non-protein in character but which contain the necessary groups will respond to the biuret test.

Protein responds positively since there are pairs of CONH groups in the molecule. A copper coordination complex with the ring structure is probably produced. Short chain polypeptides give a pinkish violet color, longer one including proteins a more purple blue. The amino acid histidine gives a pink color, which depends on the peptide linkage, does not vary greatly in intensity from protein to protein. Several procedure based on this method have been suggested for quantitative determination of milk proteins, but it is not in general use in dairy research.

19.2.5.2 Materials

1. 10% NaOH
2. 0.5% CuSO₄

19.2.5.3 Method

To 2 to 3 ml of test solution in a test tube add an equal volume of 10% sodium hydroxide solution, mix thoroughly, and add a 0.5% copper sulphate solution drop by drop, mixing between drops, until a purplish-violet or pinkish-violet color is produced. The color depends upon the nature of the protein, proteoses and peptones give a decided pink; the color produced with gelatin is not far removed from a blue.

19.2.6. Ninhydrin reaction

19.2.6.1 Principle

This test gives positive results with proteins, peptones, peptides, amino acids and other primary amines, including ammonia. Proline and hydroxyproline give yellow color with ninhydrin, while other acids give blue to purple color.

19.2.6.2 Materials

1. 0.1% Ninhydrin
2. pH paper

19.2.6.3 Method

To 5 ml of dilute test solution, which must be approximately between pH 5 and pH 7 (a few drops of pyridine or a few crystals of sodium acetate may be used to adjust the pH), add 0.5 ml of 0.1 % ninhydrin, heat to boiling for one to two minutes, and allow to cool. A blue color develops if the test is positive.

19.2.7 Folin test

A phosphomolybdotungstic acid reagent designed by Folin for phenol has been widely used for detection and analysis of indolyl and phenol groups in amino acids. A characteristic blue color is formed when amino acid solution is warmed with this reagent. The color so formed is due to the reaction of alkaline copper with the amino acid and the reduction of phosphomolybdate by tyrosine and tryptophan present.

19.2.7.1 Materials

1. Alkaline Na₂CO₃ solution (2% in 0.1 N NaOH)
2. CuSO₄-Na; K tartarate solution (0.5 % CuSO₄) in 1 % Na, K tartarate) prepared fresh by mixing stock solutions.
3. “Alkaline solution” (prepared by mixing 50 ml of the reagent (1) and 1 ml of the reagent (2) ).
4. Folin-Ciocalteau reagent

19.2.7.2 Method

Add 5ml of the alkaline solution to 1 ml of the test solution. Mix thoroughly and allow to stand at room temperature for 10 mins. Add 0.5 ml diluted Folin-Ciocalteau reagent rapidly with immediate mixing. Observe for development of color after 30 mins. Development of characteristic blue color indicates presence of indolyl or phenol group.

19.2.8 Sakaguchi test

19.2.8.1 Principle

Arginine and other guanidyl derivatives (glycocyamine, methylgyanidine etc) react with hypo bromide and alpha napthol to give a red colored product.

19.2.8.2 Materials

1. Sodium hydroxide solution (40%)
2. Alpha napthol solution (1% in alcohol)
3. Bromine water (a few drops of bromine in 100 ml distilled water)

19.2.8.3 Method

Mix 1 ml of sodium hydroxide with 3 ml of test solution and add 2 drops of alpha napthol. Mix thoroughly and add 4 to 5 drops of bromine water. Note the color formed. Formation of a red color indicates presence of guanidine group. This is a very sensitive and specific test.

19.2.9 Nitroprusside test

19.2.9.1 Principle

Sodium nitroprusside reacts with compounds containing sulphahydryl groups produce an intensely red but somewhat unstable color.

19.2.9.2 Materials

1. Sulphur amino acids (1.0% cystine, cysteine and methionine)
2. Sodium nitroprusside (2% prepared fresh)
3. Ammonium hydroxide

19.2.9.3 Method

Mix 0.5 ml of a fresh solution of sodium nitropruside with 2 ml of the test solution and add 0.5 ml of ammonium hydroxide.

19.2.10 Spectrophometric method

The use of infrared and ultraviolet spectra offers a mean of identification of amino acids and their derivatives. Spectrophometric method of detecting and determining amino acids in the intact protein offer advantages over chemical methods in that they do not involve hydrolysis; which often leads to partial decomposition of some amino acids.

The absorption of UV radiation at wave lengths of 280 nm, can be used as a method for detecting and determining some amino acids content. The light is absorbed by the amino acids tyrosine, tryptophan and phenylalanine.