Proteins are made up of amino acid residues joined by peptide bonds. Due to their polypeptide structure and different amino acid residues, protein reacts with a variety of reagents to form coloured products. These tests, known as colour reactions of proteins, are of importance in qualitative detection and quantitative estimation of proteins, and of their constituent amino acids in body fluids and other biological materials.
Proteins and amino acids used in different experiments:
- Egg albumin is an egg protein, which is sx2oluble in water.
- Casein is the major protein in milk. It is a phosphoprotein with phosphate groups attached to the hydroxyl groups or serine and threonine residues. It is deficient in cysteine.
- Gelatin is formed from collagen, the connective tissue protein, by boiling with water. It is a rich source of amino acid glycine. It is deficient in tyrosine, tryptophan and cysteine.
- Metaproteins, proteoses and peptones are partially hydrolysed products of proteins like albumins and globulins. Albumin has relatively low molecular weight. Gelatin, metaproteins, proteoses and peptones are derived proteins.
EXPERIMENT 1. COLOUR REACTIONS OF PROTEINS BASED ON PEPTIDE BONDS AND TYPE OF AMINO ACID RESIDUES
Solution: 10% Egg-white or albumin
Biuret Reaction
Experiment | Observation | Inference |
|---|---|---|
To 2 ml of sample solution, add 2 ml 5% NaOH and 3 drops of 1% CuSO4. Repeat the test with distilled water (control) | Purple-violet or pink colour in test Blue colour in control | Peptide linkages present |
- The reaction is so named since biuret (NH2-CO-NH-CO-NH2) formed by the condensation of two molecules of urea when heated. CO-NH is the peptide linkage in biuret. At least two peptide bonds in the molecule are required for a positive test. Individual amino acids and dipeptides will not answer this test.
- CuSO4 is converted to Cu(OH)2 which chelates with peptide linkage in proteins to give the colour.
- The colour varies depending on the number of peptide linkages; albumin/ globulin give violet, proteoses purple and peptones dark pink colour indicating that albumin/ globulins have largest number of peptide linkages and peptones the least.
- This reaction can be used for quantitative estimation of proteins.
Ninhydrin Reaction
Experiment | Observation | Inference |
|---|---|---|
To 1 ml of sample solution, add 0.1% ninhydrin solution, boil and cool | Ruhemann purple colour | Amino acid present |
- All α-amino acids give purple colour. The imino acids, proline and hydroxyproline give yellow colour. The coloured complex is known as Ruhemann's purple. Glutamine and asparagine produce brown colour.
- α-amino acid + ninhydrin → aldehyde + hydrindantin + NH3 + CO2; hydrindantin + NH3 + ninhydrin → Ruhemann's purple + 3H2O.
- Proteins will give a faint blue colour.
- This reaction is often used to detect amino acids in chromatography.
- Proteins do not give a true colour reaction; but N-terminal amino group of a protein can react with ninhydrin to produce a faint blue colour.
Xanthoproteic Reaction (for Aromatic Amino Acids)
Experiment | Observation | Inference |
|---|---|---|
To 2 ml of sample solution, add 1 ml conc HNO3 and boil | Yellow precipitate | Aromatic amino acids, i.e. tyrosine, tryptophan or phenylalanine present |
Cool test tube and add 40% NaOH excess | Colour of precipitate and the solution change to orange |
Yellow colour is due to the formation of nitro derivatives of benzene ring containing amino acids (tyrosine and tryptophan), the colour turns orange due to ionization when alkali is added. All proteins usually respond to this test. This reaction is also the basis of yellow stain in skin by nitric acid. Nitration of phenylalanine under these conditions normally does not take place.
Modified Millon's Reaction (Cole's Test)
Experiment | Observation | Inference |
|---|---|---|
To 2 ml of sample solution, add 2 ml 10% HgSO4 in 10% H2SO4, boil, add 5 drops of 1% sodium nitrite, heat gently | Red PPT of mercury phenolate | Tyrosine present |
- The colour is due to the formation of nitrated mercury phenolate ion of tyrosine (hydroxyphenyl group) present in proteins.
- Heat coagulable proteins give red PPT, whereas smaller molecules of proteins like peptones give red coloured solution without PPT.
- Gelatin and tapioca both are poor in tyrosine, does not give the test.
- Chloride interferes with this reaction; so it is not suitable for urine test.
Aldehyde Test for Indole Nucleus (Hopkins-Cole's Test)
Experiment | Observation | Inference |
|---|---|---|
To 2 ml of sample solution, add 1 ml of 1:500 (0.2%) formalin, 1 drop 10% HgSO4 in 10% H2SO4. Add 2 ml conc. H2SO4 slowly and carefully along the side of the test tube. Do not mix | Violet ring at the junction of two liquid layers due to indole ring | Tryptophan present |
- Mercuric sulphate cause mild oxidation of indole group of tryptophan, which condenses with an aldehyde to give the coloured complex
- p-Dimehylaminobenzaldehyde and strong hydrochloric acid (Ehrlich's reagent) give dark blue colour.
- Gelatin, poor in tryptophan, does not give the test.
Sakaguchi Test for Guanidine Group (Reaction of Arginine)
Experiment | Observation | Inference |
|---|---|---|
To 2 ml of sample solution add 2 drops of 1% α-naphthol in alcohol, 4 drops of 40% NaOH, and 8–10 drops of bromine water | Bright red colour due to guanidium group | Arginine present |
- Instead of NaOH and bromine water, 8 to 10 drops of alkaline hypobromide (NaOBr) can be used as a single reagent.
- Guanidino groups in arginyl residues of proteins react with the α-naphthol and NaOBr to give the coloured complex.
- This test is given by albumin, globulin and gelatin as it contains arginine.
Test for Sulphur-containing Amino Acids
Experiment | Observation | Inference |
|---|---|---|
To 2 ml of sample solution add 2 ml 40% NaOH, boil for 3 minutes, cool, and add 2–3 drops of lead acetate | Black or brown PPT | Cysteine or cystine present |
- Avoid excess of lead acetate solutions, which will form white PPT.
- Organic sulphur in cysteine and cystine are released as inorganic S2- ions which form lead sulphide as follows:
- Methionine does not give this test as the sulphur group in this amino acid is in thioether linkage, which is difficult to break, and not released by treatment with NaOH. Albumin and keratin will answer this test, but casein (containing methionine) will not.
Pauly's Test for Imidazole Group and Phenolic Hydroxyl Group
Experiment | Observation | Inference |
|---|---|---|
To 0.5 ml of 0.5% sulphanilic acid add 0.5 ml 1% NaNO2, mix, wait for 1 min, add 1 ml of sample solution | Cherry red colour | Histidine present |
Then add 1 ml of 10% Na2CO3 | Orange red colour | Tyrosine present |
6Diazobenzene sulphonic acid reacts with the imidazole ring of histidine or the phenolic hydroxyl group of tyrosine to give the coloured products in the alkaline medium.
Test for Phosphoprotein (Neumann's Test) (Test with Casein Solution)
Experiment | Observation | Inference |
|---|---|---|
To 5 ml of sample (casein) solution add 2 drops of chlorophenol red indicator | Dark pink colour | pH > 5.4 |
Step I | ||
Add 1% acetic acid drop by drop Decant the supernatant leaving only the precipitated casein in test tube | Yellow colour with maximum PPT | pH = 4.6, which is the isoelectric pH of casein |
Step II | ||
Add 12 drops of conc H2SO4, 4 drops of conc HNO3. Heat the test tube continuously and slowly shaking it with caution | Contents of test tube char and turn brown or black | |
Step III | ||
After no brown fumes are seen in the test tube, add 3 more drops of conc. HNO3 and heat | Colour of digest changes to orange | |
Step IV | ||
Repeat step II, 2 or 3 times until the liquid and fumes turn colourless After cooling, add 5 ml ammonium molybdate | Colour of digest changes from orange to yellow to colourless Very fine canary yellow PPT | Organic phosphorous is converted to inorganic form (PO43−) Phosphorous is present in casein, it is a phosphoprotein |
Air-cool the test tube keeping on test tube rack, not in cold water.
Phosphorous bound with casein is released as inorganic phosphate by digesting with conc H2SO4 and conc HNO3. This inorganic phosphate reacts with ammonium molybdate to produce canary yellow precipitate.