Practical Biochemistry for MBBS, MD Biochemistry

1. COLOUR
REACTIONS OF
PROTEINS
PROF (DR) VIYATPRAJNA ACHARYA
KIMS & PBH, BHUBANESWAR, ODISHA

2. WHY THESE
TESTS??
 TO IDENTIFY PROTEINS OR AMINO
ACIDS
2 TESTS ARE FOR PROTEINS, REST
ARE FOR IDENTIFYING AMINO ACIDS
BED-SIDE TESTS/ IN CLINICS
A. COLOUR REACTIONS
B. PRECIPITATION REACTIONS
C. REACTIONS OF SPECIFIC PROTEINS
D. REACTIONS OF SPECIFIC AMINO ACIDS

3. GENERAL TEST FOR PROTEINS
BIURET TEST
• Biuret- 2 urea molecules when join together on
heating at 1550c
• Test comes positive with 2 or more peptide
linkages

4. WHY & HOW- PRINCIPLE & PROCEDURE
• Principle: cupric ion in an alkaline medium forms a violet
coloured complex with peptide bond nitrogen of peptides
and proteins. The minimum requirement for a positive test
is presence of at least two peptide bonds in the molecule.
• Procedure: To 2 ml of sample solution, add 2 ml of 5%
sodium hydroxide and 3 drops of 1% copper sulfate. A
purplish violet colour is obtained.
• Repeat the test with water as control.

5. Experiment Observation Inference
1. Biuret Test
To 2ml of sample
solution 2 ml of 5%
sodium hydroxide
and 3 drops of 1%
copper sulphate was
added.
The test was
repeated with water
as control.
In the sample test
tube a deep purple
colour developed and
in the control tube
blue colour was
there.
The sample contains
a tripeptide, a
polypeptide or a
protein

6. XANTHOPROTEIC TEST
• Xanthos- yellow
• Test for phenyl group present in protein-
• Principle: The benzene ring of Tyrosine and Tryptophan undergo nitration on treatment with
strong nitric acid in higher temperature. Nitration of Phenylalanine under these conditions normally
doesn’t take place. The nitrated derivatives are of yellow colour which turns orange when made
alkaline.
• Procedure: To 2ml of sample solution add 1 ml of concentrated nitric acid. A white precipitate is
obtained due to denaturation of proteins. Heat the solution for 1 minute and cool under tap water.
Note the yellow colour obtained. Add 5 ml or more of 40% sodium hydroxide solution. The yellow
colour deepens to an orange colour.

7. ROSENHEIM’S TEST
• Acree-Rosenheim reaction- Tryptophan
• Principle: This test is specific for Tryptophan amino
acid. Mercuric sulfate causes mild oxidation of
indole group of Tryptophan, which condenses with
an aldehyde to give a violet coloured complex.
• Procedure: To 1 ml of protein solution add 1 drop of
1:500 formaldehyde and then 1 drop of 10%
mercuric sulfate in 10% Sulphuric acid. Mix well and
gently suspend 2 ml of concentrated Sulphuric acid
along the sides of the test tube. A violet ring
develops at the interface.

8. NINHYDRIN TEST
• Principle: ninhydrin reacts with α- amino groups
of proteins and free amino acids to give a blue or
purple coloured complex. This is answered by all
proteins, peptones, peptides, amino acids and
other primary amines.
• α- amino acids react with ninhydrin to form
aldehyde and hydrindantin. Hydrindantin in turn
reacts with ninhydrin to form the blue-coloured
complex.
• Procedure: To 1 ml of protein solution add 2
drops of 0.1% Ninhydrin solution. Boil for 1-2
minutes and allow to cool. A blue colour develops
if the test is positive.
• Proline and Hydroxyproline give a yellow
colour.

9. MOLISCH’S TEST
• Principle: proteins containing carbohydrates as
the prosthetic group (glycoprotein) e.g. Albumin,
which contain bound carbohydrates, answer this
test.
• Procedure: To 2 ml of albumin solution add 5
drops of Molisch’s reagent, mix and carefully
suspend 2 ml of concentrated Sulphuric acid. A
purple ring is formed at the interface.