Color

Reaction
What are the building units of the protein ?
Basic structure of the Amino acid
Peptide bond formation
 Color Reaction Tests

Heat Lead Sulfide

Biuret Test Millon’s test
Coagulation test
 Protein Samples

Albumin Casein

Egg white Milk protein

Gelatin

Animal source
Biuret test Principle

• A Biuret test is a chemical test used to determine the presence of a peptide bond in a substance.
It is based on the biuret reaction in which a peptide structure containing at least two peptide
links produces a violet color when treated with alkaline copper sulfate. in presence of an
alkaline solution
• The deeper the purple color, the higher the number of peptide-copper complexes.
1. Biuret Test

1 ml protein
+ 1 ml NaOH Violet Color : Albumin, Casein, Gelatin
+ 2 dps CuSO4
 No change in color Formation of purple color

(The unknown is not a protein ) (The unknown is protein )

Heat Coagulation Test Princple
The principle of heat coagulation test is the change in the structure of proteins as a result of heat.
Heating a protein results in denaturation of protein due to the breaking of certain bonds responsible
for the tertiary and quaternary structure of proteins to form an insoluble mass. The mass formed
doesn’t dissolve back to the liquid.
2 . Heat Coagulation Test

Permanent Coagulation: Albumin

2 ml protein

No Permanent Coagulation: Casein or Gelatin

 Heat Coagulation test

Permanent frothing + Coagulation No Permanent frothing + No Coagulation

(Albumin) (May be casein or gelatin)

Millon’s Test Principle
• Millon’s test is an analytical test used for the detection of the amino acid tyrosine, which is the
only amino acid containing the phenol group.
• Millon’s test is based on the principle of nitrification of the phenol group in tyrosine, which then
forms complexes with heavy metals like mercury. The reagent used for the test is called Millon’s
reagent, and it consists of mercuric nitrate and mercurous nitrate that is dissolved in concentrated
nitric acid. In the test, the phenol group on the tyrosine molecule is nitrated by the nitric acid
present in the reagent. The nitrated tyrosine then combines with the mercury ions in the solution to
form a red or pink colored precipitate or solution.
2. Millon’s Test

Pink Coagulum : Albumin, Casein

1 ml protein
+ 3-5 dps Millon’s
reagent

No Pink Coagulum or Solution : Gelatin

 Millon’s test

Pink Coagulum No Pink Coagulum

It may be Casein It may be gelatin
Reduced Sulfur Test Principle
• Lead sulfide test (or Lead acetate test) is a biochemical test for the detection of amino acids
containing sulfur, S-S group in cysteine, and S-H group in cystine.
• Reaction conditions release sulfur from these amino acids, sulfur reacts with the lead acetate
and forms lead sulfide, which results in a black residue.
 Reduced Sulphur
Test

Black deposit Black turbidity No black deposit

It is Albumin It is Casein It is Gelatin

Color Reaction Sheet

Conclusion: