Chemical test for protein

Requirements:

 
Glassware: Test tube, test tube stand, beaker, test tube holder, glass rod, funnel, pipette, wash bottle, wire gauze, burner.

Chemical: sodium hydroxide, copper sulphate, ninhydrine solution, nitric acid, ammonium hydroxide, mercuric sulphate, lead acetate, glacial acetic acid, formaline solution, ammonium molybodate, sulphuric acid, sodium nitrate.

Theory  for Chemical test

Amino acid- Amino acid are the monomer unit or building block of the biopolymer protein amino acid contains both amino and carboxylic acid functional group attach acid are present in nature , in which only 20 are present in protein form all from of life plant, animal or microbial. Essential amino acid- those amino acid which are not synthesized in our body are called essential amino acid like histadine, isoleucine. 

Non essential amino acid- those amino acid which are synthesized in our body are called non essential amino acid like aspargin, aspartic acid, glutamic acid. Proteins are high molecular weight compound made up of number of amino acid. protein are also be defined as high molecular weight polymer of   amino acid joined together with peptide bond .protein is the polymerized form of peptide. Proteins are classified in three groups namely simple, conjugatedand derived protein. 

Simple proteins are also classified into sub group- albumin, glutelins, albuminoids, histones, prolamines, globulin, globin protamine. Conjugated proteins are classified as: nucleoprotein, metalloprotein, lipoprotein, phosphoprotein, glycoprotein, chromoprotein. Structures of protein are classified as: primary structure secondary, tertiary, quaternary structure.

Procedure  for Chemical test for protein

Different different chemical test for protein is given below-

Biuret Test

Take two ml sample solution in test tube. Add two ml sodium hydroxide solution. Mix well then add drop wise copper sulphate solution and mix. Purplish violet color appears that indicates presence of protein.

Ninhydrin test

Take two mL sample solution in test tube then add 0.5 mL ninhydrin solution.  Heat the mixture up to boiling and cool. If blue colour appears that confirm protein is present.

Haller’s test

Take two mL concentrate nitric acid in test tube then inclined the test tube and add two mL sample solution through wall of test tube. White precipitate appeared at junction of two fluids indicates presence of protein.

Xenthoprotic test

Take two mL sample solution in test tube followed by addition of 1 mL concentrate nitric acid. A white precipitate appears then heat the mixture until solution become yellow.  Cool it. Add ammonium hydroxide solution in excess. Appearance of orange colour indicates the presence of aromatic amino acid.

Millan-Nasse’s test

Take three mL of sample solution in test tube then add mercuric sulphate.  Mixed well then boil for two minutes then cool and add sodium nitrite sulphate. Appearance deep red colore confirms the tyrocin amino acid.

Lead sulphide test or Lead blackening test

Take two mL sample solution in a test tube and add two mL sodium hydroxide solution, mix well and boil for one minute then add five drop lead acetate solution to the above mixture. Brown colored precipitate confirms the presence of cysteine.

Lead acetate test

Add two mL of sample solution a test tube containing two mL sodium hydroxide solution. Then heat up to boil and cool it.  Add little glacial acetic acid followed by one mL lead acetate solution. Precipitations of blackish precipitate indicate the presence of sulpher containing amino acid. 

Aldehyde test

Take three mL sample solution in a test tube followed by addition of two mL formaline solution. Then add two drop mercuric sulphate and mix properly. Inclined the test tube and add two mL concentrate sulphuric acid with the side of wall of test tube. Appearances of reddish violet color indicate the presence of tryptophan amino acid.

Neumann’s test

Take three mL of sample solution in test tube; add five drops sulphuric acid followed by three drops concentrate nitric acid. Mix well and heat the mixture until solution becomes colorless then add one mL concentrate nitric acid and one mL ammonium molybodate and heat the content. Cannary yellow colours appear, that confirms presence of casein protein.

Result: In the given sample solution protein (aromatic amino acid, tyrocin, cestine, tryptophan, and casein) is present. It is confirmed by the different test such as Biuret test,Ninhydrin test, Haller’s test, Xenthoprotic test, Millon-Nasse’s test, Lead sulphide test, Lead acetate test, Aldehyde test, Neumann’s test.