Amino Acid + Amino Acid + Amino Acid Peptide linkage Proteins (many peptide linkages)

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3 Amino Acid + Amino Acid + Amino Acid Peptide linkage Proteins (many peptide linkages)

4 Proteins Casein Gelatin Albumin Peptone Amino Acids Cysteine Arginine Tryptophan Tyrosine

5 Cysteine Tyrosine Arginine Tryptophan

6 Tests to be done: Ninhydrin s test (specific to all Proteins & Amino acids) Biuret s test (specific to Proteins) Sakaguchi s test (specific to Arginine) Nitropruside s test (specific to Cysteine) Millon s test (specific to Tyrosine) Hopkins-Cole s test (specific to Tryptophan)

7 Ninhydrin s test Ninhydrin reaction Principle: Ninhydrin is Specific for Amino Acids & Proteins to differentiate between Carbohydrates (-ve) and Amino Acids & Proteins (+ve). Ninhydrin Reacts with α-amino acids ( NH 2 ) in proteins giving a purple colored complex, except Proline and hydroxy proline gives yellow color(no NH 2 ). Ninhydrin is most commonly used as a forensic chemical to detect fingerprints, as amines left over from proteins sloughed off in fingerprints react with ninhydrin giving a characteristic purple color. Procedure & observation: To 1 ml amino acid solution in a test tube, add 2 drops of ninhydrin reagent. On cold and observe the formation of a purple color, OR - Put in a boiling water bath and observe the formation of a purple color.

8 Ninhydrin s test Few drops of B Mix Mix Heat 2-3 min 1ml of protein solution Mix C D Result Unknown A Proteins or Amino Acids & Carbohydrates Reagent B Drops of Ninhydrin reagent Observation C Dark purple colour on heat Proteins (free NH 2 ) D Purple-Violet colour or ppt on cold Amino Acids(free NH 2 ) D No reaction Carbohydrates

9 Biuret s test Principle: Biuret test is Specific for Proteins To differentiate between Proteins (+ve) and Amino Acids (-ve). The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in proteins to form a blue to violet complex known as the Biuret complex. Two peptide bonds at least are required for the formation of this complex. Procedure & observation: To 1 ml of protein solution in a test tube, add 2 ml of 10% sodium hydroxide solution and 2 drops of 1% copper sulfate solution. Mix well; a violet color is obtained with albumin, casein & gelatin and a pinkish violet color with peptone.

10 Biuret s test 1ml of NaOH 2 drops of B Mix Mix 1ml of protein solution C Unknown A Proteins or Amino Acids Reagent B 2 ml NaOH, then 2 drops of CuSO4 (1%) Result Observation C Violet colour Proteins (reacts with Di-peptide bonds) C No change in colour (Blue) Amino Acids (No di-peptide linkage)

11 Sakaguchi s test Principle: Sakaguchi test is Specific for Arginine Sakaguchi's test is positive for the amino acid containing the guanidine group in Arginine. Guanidine group present in the amino acid reacts with α-naphthol and alkaline hypobromite to give red-coloured complex. Procedure & observation: To 1 ml of protein solution in a test tube, add 40%NaOH (2-4 drop) + Ethanolic a-naphthol (2 drops) and Bromine water (5-10 drops) Mix well; a red-colour complex will be formed with Arginine or protein containing Arginine.

12 Sakaguchi s test Few drops of B Mix Mix 1ml of protein solution C Unknown A Proteins or Amino Acids Reagent B 2 drops NaOH + 2 drops Alcoholic α- naphthol. Mix then add Bromine water (for safety, use Chlorine water) Result Observation C Red colour Proteins containing Arginine C Red colour Arginine (Guanidine gp)

13 Nitroprusside s test Principle: It is specific for Proteins containing sulfur, -SH (in cysteine & cystine) give a red-purple colour called Mörner test. Procedure & observation: To 1 ml of protein solution containing Cysteine/Cystine in a test tube, add 3 drops of a 5% solution of sodium nitroprusside - Mix well and add few drops of ammonia solution, a deep red-purple color appears; called also Mörner test. l

14 Nitroprusside s test Few drops of B Mix Mix 1ml of protein solution C Unknown A Cystine or Cysteine in Proteins or Amino Acids Reagent B 3 drops Sodiun nitropruside Then, 1 ml Ammonia soln. Result Observation C Red purple colour Amino Acid: Cystine or Cysteine (-SH gp) C Red purple colour Proteins containing Cystine or Cysteine (-SH gp)

15 Millon s test Principle: It is specific for Tyrosine. Millon's reagent (Hg/HNO3) gives positive results with proteins containing the phenolic amino acid tyrosine. Procedure & observation: To 1 ml of protein solution in a test tube, add 1ml of Millon s reagent [Hg(SO 4 ) + HNO 3 ] then add NaNO 2 - Heat up for few min A red pink colour appears

16 Millon s test Few drops of B Mix Mix Heat 2-3 min 1ml of protein solution C Unknown A Tyrosin in Proteins or Amino Acids Reagent B 1 ml Millon reagent [Hg(SO 4 ) + HNO 3 ] then heat Result Observation C Red-Pink colour Amino Acid: Tyrosin (-Ph-OH gp) C Red-Pink colour Proteins containing Tyrosin (-Ph-OH gp)

17 Aldehyde test Principle: It is Specific for Tryptophan. Sulphuric acid in presence of mercuric sulphate oxidizes the indole nucleus of tryptophan. The product formed reacts with aldehydes to form violet colored complex. Procedure & observation: To 1 ml of protein solution in a test tube, add 2 drops of 0.2 of Formalin + 1 drop of 10% HgSO 4 ] and mix well. Incline the test tube and slowly add 2 ml of concentrated H 2 SO 4 on the inner wall of the test tube to form violet-purple ring at the junction of the 2 layers. A reddish violet ring is formed at the junction between the 2 layers with albumin and casein; gelatin gives negative results.

18 Aldehyde test Few drops of B 2 ml of H 2 SO 4 On the side wall Tryptophan Mix Mix 1ml of protein solution C H 2SO 4 Unknown A Tryptophan in Proteins or Amino Acids Reagent B [Formalin + HgSO 4 ] then add conc H 2 SO 4 Result Observation C Violet/Purple colour ring Amino Acid: Tryptophan (Indole ring) C Violet/Purple colour ring Proteins containing Tryptophan (Indole ring)

19 Some additional tests for qualitative identification of proteins A) Biuret Test: Reagents: 1) 1 % CuSO4 2) 5% NaOH Procedure: To 1 ml of proteins solution, add 2ml of 5% NaOH and 2 drops of 1% CuSO4 solution, mix well. A violet color forms, which indicates the presence of peptide bonds in the molecule. B) Heat coagulation test Place about 5 ml of egg-white solution (albumin solution) in a test tube and heat the top part of the solution only. Note that it gradually becomes cloudy and a flocculent precipitate of coagulated protein is produced.

20 C) Xanthoproteic Test: Reagents: a- Concentrated HNO3 b- 40% NaOH Procedure: a. To 3 ml of protein solution, add 1 ml of conc. HNO 3. Mix and boil for 1min. Cool under tap water. Yellow color forms. b. Add 2 ml of 40% NaOH to make the solution alkaline. The color changes to orange indicating the presence of aromatic amino acids. Principle: This test is specific to the amino acids containing the benzene ring (aromatic amino acids). Phenylalanine, tyrosine and tryptophan reacts with conc. HNO3 at high temperature to form nitro-compounds which are yellow in color, it turns to orange color in an alkaline medium. D) Picric acid test: To 3 ml of gelatin solution in a test tube, add 2ml of saturated picric acid solution, a yellow gelatinous precipitate is formed.

21 Identification of unknown protein solution Biuret Test Violet Color No Violet Color Solution is protein Solution is not protein (carbohydrates or amino acids) Heat coagulation test Cloudness at top part No Cloudness at top part of the solution of the solution (Albumin) (Casein-Gelatin-Peptone) Reaction to litmus paper Neutral Soln. is (peptone or Gelatin) Picric acid Test Alkaline Soln. is (Casein) No yellow gelatinous ppt. Yellow gelatinous ppt. Soln. is (Peptone) Soln. is (Gelatin) (Peptone gives pink color with biuret)

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