1. COLOUR REACTIONS OF PROTEINS (ALBUMIN) - DR. SHASHIKALA ASSISTANT PROFESSOR - MR. SHIVANAND TUTOR

2. COLOUR REACTIONS OF PROTEIN (ALBUMIN) Proteins are high molecular weight and condensed products of amino acids held together by peptide bonds. Because of their peptide structure and due to presence of different functional groups, amino acids react with variety of reagents to form coloured products.

3. Albumin (Latin, albus = white) It is globular protein with one polypeptide chain with 585 amino acids. It has a molecular weight of 69,000 D. Albumin is a plasma protien synthesized by hepatocytes in liver & constitutes the major part of plasma proteins. Half-life of albumin is about 20 days. Liver produces about 12 g of albumin per day, representing about 25% of total hepatic protein synthesis. Helps in maintainance of Colloidal Osmotic Pressure of Plasma, Transport of many compounds (such as Bilirubin, non-esterified fatty acids, Drugs, Hormones, Metals etc..,). Besides this albumin has a maximum buffering capacity.

4. 1. Biuret test ExperimentObservationInference Take 4ml of sodium hydroxide solution in a test tube, add 5 drops of copper sulphate solution and mix well. Divide solution into 2 parts To first part, add 1ml of albumin solution To second part add 1ml of distilled water (control) A violet color No violet color Albumin is a protein, contains more than two peptide bonds

5. Principle Alkaline copper sulphate reacts with compound containing two or more peptide bonds to give violet colored complex. This test requires minimum two peptide bonds to give positive results. Free amino acids do not answer this test. Intensity of color developed is a measure of number of peptide bonds present in the protein.

6. 2. Xanthoproteic test ExperimentObservationInference 0.5ml of con.nitric acid is added to about 1ml of protein solution and boiled for three minutes. Cooled and 3ml of 40% NaOH is added to make the solution strongly alkaline A yellow color is formed which turns bright orange with alkali albumin contains aromatic amino acids

7. Principle Amino acids that contain substituted aromatic nucleus form yellow nitroderivatives on heating with con.nitric acid. On addition of alkali, the nitrophenyl group get ionized giving deep yellow (orange) colour to the solution. This test for amino acids containing benzene ring such as tyrosine, tryptophan and phenylalanine.

8. 3. Millon’s test ExperimentObservationInference 1ml of Millon’s reagent is added to 1ml of protein solution and boiled for 2 minutes, then cool to room temperature and 5 drops of sodium nitrite is added A red colored complex or a red coagulum is formed The given protein contains hydroxy benzene ring containing amino acid (Tyrosine)

9. Principle Compounds containing hydroxy-benzene ring react with Millon’s reagent to form a red colored complex. Phenolic compound tyrosine gives positive reaction. Reagents- Million’s reagent:10% mercuric sulphate in 10% H 2 SO 4 and 5% sodium nitrite solution.

10. 4.Hopkin’s Cole test ExperimentObservationInference 2ml of protein is taken in a test tube and 2ml of Hopkin Cole reagent is added and mixed. 2ml of con.sulphuric acid is now added slowly along the sides of test tube Reddish violet ring is formed at junction of two liqiuds Given protein contains amino acid with indole group (tryptophan)

11. Principle Aldehyde react with the oxidized products of indole nucleus of tryptophan to give violet coloured (reddish violet) ring. Con.sulphuric acid behaves as oxidizing reagent here. Color produces is more intense if the reaction is carried out at low temperatures.

12. 5. Sakaguchi test ExperimentObservationInference To 1ml of protein solution, 2 drops of Molisch’s reagent is added and mixed well. 1ml of alkaline hypobromide solution is added A carmine red color is produced The given protein contains amino acid with guanido group (arginine)

13. Principle The guanido group in presence of alpha napthol and an oxidizing agent such as alkaline hypobromite gives a carmine red color. Reagents- 1g of alpha-naphthol in 100ml of 95% alcohol (Molisch’s reagent) & alkaline hypobromite (40% NaOH with bromine water).

14. 6. Pauly-diazo test Experiment Inference Observation In Inference Observation Inference ExperimentObservationInference To 1ml of 0.5% of sulphanalic acid and equal volume of 0.5% of sodium nitrite is added along with1ml of protein solution. To this 1ml 10% sodium carbonate is added to make alkaline A wine red coloured complex is formed Given protein contains imidazole group of histidine

15. Principle Test is for detection of histidine. Diazotised sulphanilic acid reacts with the imidazole ring of histidine to form wine red coloured complex.

16. 7. Lead acetate test ExperimentObservationInference To 2ml of albumin solution add 2ml of 40% NaOH and boil for 2- 3mins and cool and add 2 drops of lead acetate solution Brown or black precipitate Albumin contains sulphur containing amino acids (cysteine and cystine)

17. Principle Amino acids cystine and cysteine are hydrolysed. Which gives sulphide ions and addition of lead acetate leads to formation of lead sulphide which is black in colour. Report: Albumin is a protein that contains aromatic aminoacids (tyrosine, tryptophan and phenylalanine), arginine, sulphur containing amino acids (cystine and cysteine) and histidine.

18. Ninhydrin test (for free α amino groups) Ninhydrin is a powerful oxidizing agent. Reacts with α amino groups of amino acids between pH 4 to 6 to give deep blue or purple coloured complex. This reaction is used for the detection and estimation of amino acids.

19. Neumann’s test (for organic phosphorus) Organic phosphate present in phospho-protein converted to in-organic phosphate on boiling with strong sodium hydroxide solution. Inorganic phosphate reacts with ammonium molybdate to form ammonium phosphomolybdate which produces canary yellow colour. Caesin is a phosphoprotein and answers neumann’s test.

20. Clinical significance of colour reactions Colour reactions are not only for the detecting presence of amino acids in proteins but they are also used for diagnosis of inborn errors of amino acid metabolism or aminoaciduria’s DisorderEnzyme absent/deficientAminoacids in urine Positive tests PhenylketonuriaPhenylalanine hydroxylasePhenylalanineXanthoproteic test Tyrosinemia -IFumaryl acetoacetate hydrolaseTyrosineXanthoproteic test Millon’s test Tyrosinemia -IITyrosine aminotransferaseTyrosineXanthoproteic test Millon’s test Tyrosinemia- Transient neonatal Para hydroxyl phenyl pyruvate hydroxylase TyrosineXanthoproteic test Millon’s test Hartnup’s diseaseDefective absorption of tryptophanTryptophanXanthoproteic test Hopkin cole’s test ArginemiaArginaseArginineSakaguchi test HistidemiaHistidaseHistidinePauly-Diazo test CystinuriaDefective absorption of cysteineCysteineLead acetate test

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