Here is a list of top fifteen experiments on photochemical tests for proteins: 1. Precipitation of proteins with heavy metals 2. Precipitation of proteins by alkaloid reagents 3. Tannic acid test 4. Heller’s test 5. Acetic acid-potassium ferrocyanide test 6. Biuret reaction 7. Ring biuret test 8. Nanthoproteic reaction for tyrosine, phenylalanine and tryptophane 9. Millon’s test for tyrosine and Others.
Contents:
- Experiment on the Precipitation of proteins with heavy metals
- Experiment on the Precipitation of proteins by alkaloid reagents
- Experiment on Tannic acid test
- Experiment on Heller’s test
- Experiment on Acetic acid-potassium ferrocyanide test
- Experiment on Biuret reaction
- Experiment on Ring biuret test
- Experiment on Nanthoproteic reaction for tyrosine, phenylalanine and tryptophane
- Experiment on Millon’s test for tyrosine
- Experiment on Aldehyde test for tryptophane
- Experiment on Glyoxalic acid test for tryptophane
- Experiment on Arginine test for arginine
- Experiment on Sulphur test for cystine and cysteine
- Experiment on Molisch’s test for carbohydrate group attached to protein molecule
- Experiment on Ninhydrin test
Experiment # 1. Precipitation of proteins with heavy metals:
Procedure:
ADVERTISEMENTS:
Take 3 cc of 5 per cent egg white solution and add 0.5 per cent ferric chloride solution drop by drop.
Result:
On addition of first drop, turbidity appears and it increases on addition of subsequent drops. If FeCl3 is added in excess, the turbidity disappears.
Experiments # 2. Precipitation of proteins by alkaloid reagents:
Procedure:
ADVERTISEMENTS:
In a test tube take 3 cc of 5 percent egg white solution and add 20 per cent sulphosalyclic acid.
Result:
White precipitate is obtained.
Experiment # 3. Tannic acid test:
Procedure:
ADVERTISEMENTS:
In a test tube, take 3 cc of 5 per cent egg white solution and then add 5 drops of freshly-prepared tannic acid solution.
Result:
Brownish and non-granular precipitate is formed.
Experiment # 4. Heller’s test:
Procedure:
Take 3 cc of concentrated HNO3 in a test tube and then add very carefully 3 cc of 5 per cent egg white solution by means of a pipette in such a manner that it forms upper layer. Mix gradually by rotating between palms.
Result:
A white ring is formed at the junction of the two solutions.
Experiment # 5. Acetic acid-potassium ferrocyanide test:
Procedure:
In a test tube take 3 cc of 5 per cent egg white solution, then add 3 drops of glacial acetic acid, and 3 drops of potassium ferrocyanide solution.
ADVERTISEMENTS:
Result:
White precipitate is obtained. The precipitation is due to the proteoses, which dissolve on boiling and reappear on cooling.
Experiment # 6. Biuret reaction:
Procedure:
Take 3 cc of 5 per cent protein solution in a test tube, add 1 cc of 40 per cent NaOH solution to make it strongly alkaline, and then add 2 drops of 1 per cent copper sulphate solution.
Result:
Violet or pink colour appears. This reaction is due to the peptide linkage and so it is positive with all proteins.
Experiment # 7. Ring biuret test:
Procedure:
Take 3 cc. of 45 per cent or even more dilute egg white solution in a test tube, add 1 cc of 40 per cent NaOH, then add by means of a pipette 1 cc of 1 per cent copper sulphate over the surface of the liquid very gently so that the fluids do not mix. Rotate gently.
Result:
A pink or violet ring is formed at the junction of the two fluids. Proteose and peptones give rose colour. Gelatin gives bluish pink or violet colour.
Experiment # 8. Nanthoproteic reaction for tyrosine, phenylalanine and tryptophane:
Procedure:
Take 3 cc of per cent egg white solution in a test tube, add 1 cc of conc. HNO3 and boil. First white precipitate is formed which changes to yellow. The liquid also becomes yellow. Cool the test tube and add excess of 40 per cent NaOH or ammonia to make it alkaline.
Result:
The yellow colour changes to orange. The proteoses and peptones do not form precipitate with HNO3 but their solution turns yellow to orange in the presence of alkali.
The precipitate is due to the formation of meta-proteins insoluble in HNO3 (nitric acid). The Yellow colour is due to nitro-compounds from the protein molecule containing benzene ring. When made alkaline, the nitro-compounds ionize freely and produce deep yellow or orange colour.
Experiment # 9: Millon’s test for tyrosine:
Procedure:
In a test tube take 3 cc of 3 per cent egg solution and 2 cc of mercuric sulphate reagent by pipette and boil cautiously for a minute. A yellowish precipitate is generally formed. Cool the tube and add 2 drops of 1 per cent NaNO2 (Sodium nitrite). Heat again.
Result:
The solution and the precipitate become red showing the presence of tyrosine.
Experiment # 10. Aldehyde test for tryptophane:
Procedure:
In a test tube take 3 cc of protein solution (5 per cent egg white), then add one drop of 0.2 per cent of 40 per cent formalin, then and 0.5 cc of mercuric sulphate reagent. Shake well and then add 2.0 cc of conc. H2SO4. Shake.
Result:
Violet or purple colour develops. Sometimes, a little heat is required for the colour to appear.
Experiment # 11. Glyoxalic acid test for tryptophane:
Procedure:
In a test tube take 3 cc of 5 per cent egg white solution for protein and add 3 cc of glyoxalic reagent. Now transfer this solution very carefully to another test tube containing 5 cc of conc. H2SO4 in such a manner that the two fluids do not mix. Rotate the tube gently.
Result:
Purplish violet colour develops at the junction of the fluids. The purple or violet colour is due to the presence of tryptophane, which forms condensation product with the aldehyde.
Experiment # 12. Arginine test for arginine:
Procedure:
In a test tube take 3 cc of 5 per cent egg white protein solution, then add 2 drops of 1 per cent a-naphthol solution, then add 1 cc of 40 per cent NaOH solution, and then add 2 drops of sodium hypo-bromide (NaOBr).
Result:
Bright red colour is obtained. This reaction is specifically meant for arginine which is present in all proteins.
Experiment # 13. Sulphur test for cystine and cysteine:
Procedure:
In a test tube take 3 cc of protein solution (5 per cent egg white), then add 5 drops of lead acetate which causes precipitation. Now add 40 per cent NaOH drop-by-drop till the precipitate dissolves. Boil.
Result:
Black or brown precipitate is formed, which shows the presence of cystine or cysteine group.
Experiment # 14. Molisch’s test for carbohydrate group attached to protein molecule:
Procedure:
In a test tube take 3 cc of 5 per cent egg white protein solution, then add 2 drops of 5 per cent alcoholic thymol; now incline the tube and gently add 3 cc of conc. H2SO4 (the acid should go by the side of the tube wall) in such a way that the fluids do not mix. Rotate the tube gently.
Result:
Purple-violet ring, at the junction of the fluids, is formed which shows the presence of carbohydrate group attached to the protein molecule.
Experiment # 15. Ninhydrin test:
Procedure:
Take 1 cc of 5 per cent egg white protein solution and add 4 drops of 0.1 per cent ninhydrin solution and boil for one minute. Cool the test tube.
Result:
Blue colour develops. The test gives positive results by all amino acids and their derivatives except proline and hydroxyl proline.