Introduction

Amino acids are organic compounds that contain amino and carboxyl group and therefore possess both basic and acidic properties. Proteins are polymer of amino acids and contain about 22 different amino acids, with the exception of proline and hydroxyproline, which strictly speaking are amino acids. General formula of an amino acid is as shown below:

 

All amino acids with the exception of glycine show optical activity due to presence of asymmetric carbon atom

Protein (Greek: Proteios, meaning holding first place) are one of the most important group of biomolecules. The quantity and type of protein shows a wide variation between not only among different organsims but also is different organs or parts of the same organism. Proteins perform very vital and diverse roles in organisms. A simple bacterial cell such as Escherichia coli contains more than 5000 different type of proteins.

QUALITATIVE TESTS FOR AMINO ACIDS AND PROTEINS

The various qualitative tests for detection of amino acids in a sample preparation are described.

A) NINHYDRIN TEST

Principle

The test due to a reaction between a -amino group of free amino acid and ninhdyrin. Ninhydrin is a powerful oxidising agent and in its presence, amino acids undergo oxidative deamination liberating ammonia, carbon dioxide, a corresponding aldehyde and reduced from of ninhydrin. The ammonia formed from a -amino group reacts with ninhydrin and its reduced product to give a blue substances diketohydrin. However, in case of imino acids like proline, a different product having a bright yellow colour is formed. Asparagine which has a free amide group reacts to give a brown coloured product. This test is also given by proteins and peptides.

Procedure and Observation

Add 2-5 drops of ninhydrin solution to 1 ml of test solution or sample preparation. Mix and keep for 5 min in boiling water bath and observe the development of a pink, purple or violet-blue color. Imino acids proline and hydroxyproline give a yellow colored complex.

B) XANTHOPROTEIC TEST

Principle

Amino acids containing an aromatic nucleus from yellow nitro derivatives on heating with HNO₃. The salts of these derivatives are orange in color. Proteins containing these amino acids give a positive response to this test.

Reaction

Procedure and observations

Add 1ml conc HNO₃ into ml of test solution. Cool it and then slowly pipette NaOH till the solutions alkaline. Note weather the mixture turns orange red in colour. Apperance of orange red colour denotes presence of aromatic amino acids.

Principle

Amino acids or compounds containing hydroxybenzene radical react with Millon’s regents which constitutes of mercurous and mercuric nitrates containing HNO₃, to form a red complex. Thus this test is specific for tyrosine.

Procedure and Observations

Add 2-5 drops of Millon’s reagent into 1 ml of test solution. Shake the contents and place the test tubes in boiling water bath. Development of a red color indicates presence of tyrosine. A positive reaction will also be obtained for proteins, which contain tyrosine.

The same experiment may be carried out a modified Millon’s reagent which contains 10% mercuric sulphate in 10% H₂SO₄. To 1 ml of test solutions, add 1 ml of modified Millon’s reagent and boil gently for 1 min. Cool under running tap water and then add one or two drops of 1 % NaNO₂ solutions and heat slightly. Appearance of red color denotes presence of tyrosine. The advantage of using modified Millon’s test lies in its being less susceptible to interference from inorganic salts.

Principle

The indole group of tryptophan reacts with glyoxylic acid in the presence of conc. H₂SO₄ to give a purple color. Glyoxylic acid is prepared by reducing oxalic acid with magnesium powder or sodium amalgam. Glacial acetic, which has been exposed to the sunlight also, contains glyoxylic acid and can thus be used for this test.

Reaction

Procedure and Observations

Add 1 ml of Hopkins-Cole test reagents to 1 ml of test solution. Mix and pour with pipette conc. H₂SO₄ along the sides of test tube. Observe for appearance blue or violet ring at the interface of the liquids, which would indicate presence of tryptophan.

Principle

This test is a general test compounds having a peptide bond. Alkaline copper sulphate reacts with compounds containing two or more peptide bonds to give a violet or pinkish coloured product which is due to formation of coordination complex of cupric ions with unshared electron pairs of peptide nitrogen and oxygen of water.

Reaction

Procedure and observations

Add 0.5 ml NaOH to 1 ml of test solutions and mix well. Then add 2-5 drops of copper sulphate and observe for the formation of pink or violet colour. Appearance of pink or violet colour shows presence of petides or proteins in the sample. Try this test with solutions of glutathione and its constituent amino acids and record the observations.

Principle

Diazotized sulphanilic acid couples with amines, phenols and imidazole to form highly coloured compounds. The diazonium compound is only formed in the cold, so all solutions must be cooled in ice before diazotization.

Reaction

Procedure and observations

Mix 1 ml of sulphanilic acid with 2 ml of the test solutions cooled in ice, add 1 ml of sodium nitrite and leave in the cold for 3 min. make the solution alkaline by the addition of 2 ml of sodium carbonate solution. Note whether any coloured products are formed or not.

Principle

This reaction is given by guanidinium compounds. The only amino acid containing the guanidine group is arginine, and this reacts with a -naphthol and an oxidizing agent such as bromine water or sodium hypochlorite to give a red coloured product. The other guanidine containing non-amino acid compounds also give this reaction.

Materials and Reagents

Procedure and observations

Mix 1 ml of NaOH with 3 ml of the amino acid or test solution and add 2 drops of a -naphthol. Mix thoroughly and add 4 to 5 drops of bromine water. Watch for the formation of red color which indicate presence of arginine or a guanidinium compound.