Proteins Practical Part I تجارب البروتينات

Proteins

Proteins are organic compounds of high molecular weight; they are formed of α-amino acids attached together by peptide linkages or peptide bonds.

Colour Reactions For Proteins

Proteins produce colour in certain reactions. These reactions are not quite specific for a protein molecule as such but are due to characteristic groups of particular amino acids present in it

Biuret test

When urea is heated it forms biuret .if a strongly alkaline solution of biuret is heated with very dilute copper sulphate a purple –violet colour is obtained.

The colour depends upon the presence of 2 or more peptide linkages. thus dipeptides and free amino acids do not give the biuret test. Only Histidine can give a positive reaction . biuret test is due to co-ordination of cupric ions with the unshared electron pairs of peptide nitrogen and the oxygen of water to form coloured co-ordination complex.

Procedure:

1 ml of protein solution (egg albumin)+ 2 ml of 10% NaOH ,shake the tube well, then add 0.5 ml of 0.2% CuSO4 → purple – violet colour .

Ninhydrine test:

Ninhydrine is a powerful oxidizing agent and causes oxidative decarboxylation of α-amino acids producing an aldehyde with one carbon less than the parent amino acids. The reduced ninhydrine hydrindanthin then reacts with  ammonia which has been liberated and one molecule of ninhydrine forming a blue – coloured compound . a molecule of CO2 is evolved indicating the presence of α-amino acid.

Procedure:

1 ml of α- amino acid solution +2 drops of 0.3% ninhydrine solution + water bath for 2 min. , then cool. →    blue colour.

Xanthoproteic test :

The aromatic amino acids such as phenylalanine, tyrosine, and tryptophan present in the protein give yellow precipitate when heated with conc.HNO3. On addition of alkali, the precipitate turns orange due to nitration of the aromatic ring. Collagen and gelatin do not give a positive reaction.

Procedure:

1 ml of protein solution + 1 ml of concentrated HNO₃ ,then shake the solution + heat on water bath until precipitate is produce then cool the solution + add 5 drops of 10% NaOH )or NH₄OH ) →    yellow PPT.

Millon's test:

This is a specific test for tyrosine and tyrosine of protein. Protein gives a white ppt. with Millon's reagent (10 % mercurous chloride in H₂SO₄) on heating. On addition of NaNO₂ the precipitate turns pink – red.

Procedure:

2 ml of protein solution + 1 ml of mercuric sulphate solution + heat on water bath for 10 min., cool the contents in tap water for 5 -10 min + 1 ml of 1% sodium nitrate. →   A deep red colour.

Indicates positive test.

Sakaguchi test:

This is a specific test for arginine of the protein. Sakaguchi reagent consists of alcoholic α -naphthol and a drop of sodium hypobromite. Guanidine group of arginine is responsible for the formation of red colour.

Procedure:

1 ml of protein solution +1 ml of 5% NaOH  + 2 drops of 1% ethanolic α -naphthol + 5 drops of bromine water or 5 drops of sodium hypobromite →    a red colour.

Hopkin – Cole test:

This is a specific test for tryptophan of the protein. Protein containing tryptophan gives this test positive. The reaction is characteristic of tryptophan. Because contain of indol group. Gelatin, collagen do not contain tryptophan and hence do not give this test positive.

Procedure:

1 ml of protein solution +1 drop of (1:500) formaldehyde + 1 drop of 10 % mercuric sulphate in 10% H₂SO₄ .Mix well+ gently add 3 ml of conc. H₂SO₄ along side of the test tube. Top gently at the junction of the two layers → a violet ring develops.

Nitroprusside test:

Protein with free-SH group of cysteine give reddish colour with sodium nitroprusside in ammoniacal solution

Procedure

1 ml of protein solution + drops of sodium nitroprusside in ammoniacal solution until   → appearance reddish colour.

Lead acetate test:

Identification of sulphur in protein molecular. This is a specific test for sulphur containing amino acids is boiled with strong alkali to split out sulphur  as sodium sulphide which reacts with lead acetate to give black ppt of pbS.

Procedure:

1 ml of protein solution + 1 ml of 40% NaOH Solution + 0.5 ml of 5% lead acetate ,then shake the solution, heat on water bath for 3-5 min. → black ppt. or a grey ppt.

 indicates a positive test

Test For Organic Phosphorus ( only with Casein):

The organic phosphate present in phosphoprotein is convert to inorganic phosphate on boiling with strong NaOH solution. Inorganic phosphorus react with ammonium molybidate to form ammonium phosphomolybidate which is canary yellow in colour.

Procedure:

3 ml of protein solution (Casein)+ 0.5 ml of 40% NaOH solution + heat strongly , then cool under tap water + 0.5 ml of concentrated HNO₃ + filter + the filtrate ,add a  pinch of solid ammonium molybidate + warm gently →  the canary yellow colour of precipitate.

Rosenhyime's Reaction:

This  is a specific test for indol group in tryptophan.

Procedure:

2 ml of protein solution +10 drops of FeCl3 solution ,then shake the tube well + 10 drops of formaldehyde + 2 ml of cnoc. H2SO4, wait for 1-3 min. → violet  ring.