Amino Acid + Amino Acid + Amino Acid Peptide linkage

Proteins (many peptide linkages)

Proteins Amino Acids

Casein CysteineCasein Cysteine

Gelatin Arginine

Albumin Tryptophan

Peptone Tyrosine

Arginine

Cysteine

Tryptophan

Tyrosine

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)�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)

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 (–NH2) in proteins giving a purple colored

complex, except Proline and hydroxy proline gives yellow color(no –NH2).

Ninhydrin’s test

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.

Ninhydrin’stest

Ninhydrin’stest

Few drops of B

Mix Mix Heat

2-3 min

1ml of protein

solutionC

2-3 min

Result

Unknown A Proteins or Amino Acids & Carbohydrates

Reagent B Drops of Ninhydrin reagent

Observation C Dark purple colour on heat Proteins (free –NH2)

D Purple-Violet colour or ppt on cold Amino Acids(free –NH2)

D No reaction Carbohydrates

Mix

D

Biuret’s testPrinciple: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 “Biuretcomplex”. 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.

Biuret’s test

2 drops of B

Mix Mix

1ml of NaOH

1ml of protein

solutionC

Result

Unknown A Proteins or Amino Acids

Reagent B 2 ml NaOH, then 2 drops of CuSO4 (1%)

Observation C Violet colour Proteins (reacts with Di-peptide bonds)

C No change in colour (Blue) Amino Acids (No di-peptide linkage)

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.

Sakaguchi’stest

Sakaguchi’stest

Few drops of B

Mix Mix

1ml of protein

solutionC

Result

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)

Observation C Red colour Proteins containing Arginine

C Red colour Arginine (Guanidine gp)

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

Nitroprusside’s test

Nitroprusside’s test

Few drops of B

Mix Mix

1ml of protein

solutionC

Result

Unknown A Cystine or Cysteine in Proteins or Amino Acids

Reagent B 3 drops Sodiun nitroprusideThen, 1 ml Ammonia soln.

Observation C Red – purple colour Amino Acid: Cystine or Cysteine (-SH gp)

C Red – purple colour Proteins containing Cystine or Cysteine (-SH gp)

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(SO4) + HNO3] then add NaNO2

- Heat up for few min

− A red – pink colour appears

Millon’s test

Few drops of B

Mix Mix Heat

2-3 min

1ml of protein

solutionC

2-3 min

Result

Unknown A Tyrosin in Proteins or Amino Acids

Reagent B 1 ml Millon reagent [Hg(SO4) + HNO3] then heat

Observation C Red-Pink colour Amino Acid: Tyrosin (-Ph-OH gp)

C Red-Pink colour Proteins containing Tyrosin (-Ph-OH gp)

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% HgSO4] and mix well.

− Incline the test tube and slowly add 2 ml of concentrated H2SO4 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.

Aldehyde test

2 mL of H2SO4

On the side wall

Mix

Few drops of B

Mix

Tryptophan

Result

Unknown A Tryptophan in Proteins or Amino Acids

Reagent B [Formalin + HgSO4] then add conc H2SO4

Observation C Violet/Purple colour ring Amino Acid: Tryptophan (Indole ring)

C Violet/Purple colour ring Proteins containing Tryptophan (Indole ring)

1ml of protein

solutionC H2SO4

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 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 testPlace 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.

C) Xanthoproteic Test:Reagents: a- Concentrated HNO3

b- 40% NaOHProcedure:a. To 3 ml of protein solution, add 1 ml of conc. HNO3. 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: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.

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 Alkaline

Soln. is (peptone or Gelatin) Soln. is (Casein)

No yellow gelatinous ppt. Yellow gelatinous ppt.

Soln. is (Peptone) Soln. is (Gelatin) (Peptone gives pink color with biuret)

Picric acid Test