Color Reactions of Proteins

COLOR REACTIONS OF PROTEINS
PURPOSE
This experiment allows students to determine the color reactions of proteins through the following
reactions which are the biuret reaction, xanthroproteic reaction, Millon’s test, Glyoxylic acid reaction .,
APPARATUS
 Bunsen burner
 Tripod
 Test tubes
 Test tube holder
MATERIALS
 2% Peptone soln
 2% Albumin soln
 10% sodium hydroxide - NaOH
 0.5% copper sulfate- CuSO4
 Conc. Nitric acid – HNO3
 Ammonium hydroxide – NH4OH
 Hopkins cole
 SULFURIC acid – H2SO4
 GLACIAL ACETIC ACID – CH3COOH
 LEAD ACETATE – Pb(CH3COOH)
 Magnesium- Mg
PROCEDURE
Biuret reaction
In a test tube, 1 ml of 2% albumin solution and 1 ml of Sodium hydroxide (NaOH) were mixed. Drop by
drop 0.5% Copper sulfate (CuSO4) were added. A violet color was obtained. The same procedure was
done with the mixture of 2% peptone solution and 1 ml of 10% NaOH. After that 0.5 % of Cu SO4 Copper
sulfate was added and a pink solution was observed.
Biuret reaction is a general test for proteins which shows a violet color indicating a long chained protein
and pink color for a short chained protein. It is an indicator for the determination of strong or weak
protein hydrolysis in an amino acid.
The presence of a long peptide linkage (-CONH) in any substance will test positive in the Biuret test
while no coloration is observed in amino acids that contain smaller peptides.
Xanthroproteic reaction
In a test tube, 1ml of 2% albumin and 0.5 ml on conc. Nitric acid (HNO3) was mixed then heated. The
solution turn yellow. After a few minutes it was cooled down and added with Ammonium hydroxide
(NH4OH) in excess which resulted the solution to turn orange.
The presence of a phenyl group (-C5H5) in a protein is indicated by the addition of Conc. HNO3. As a
result, proteins turn yellow and change to orange when added with an alkali (NH4OH). Examples of
amino acids are tyrosine, tryptophan and phenylalanine. These amino acids change color due to the
nitration of the benzene ring. Nitration is the addition of nitrogen to a substance.
Millon’s regaent
Millon’s regeant is prepared by mixing one part by weight of mercury with two parts by weight of conc.
Nitric acid and dilute the resulting solution with two volumes of water.
In a test tube, a 1 ml of 2% albumin solution was added and a drop of Millon’s regeant. It was then
mixed and heated.
When adding a protein to the millon’s regeant solution the protein is precipitated as mercury salt. When
it was heated, the precipitate turned into a red flocculent color and if proteins containing tyrosine it is
present. The reaction is due to the phenol group that contained the tyrosine, therefore it indicates that
the Millon’s Test is positive.
Glyoxylic acid reaction
The Hopkins-cole reagent is prepared by mixing 10 grams of powdered magnesium with water. Then
added slowly with 250 ml cold water and shaked. The mixture was filtered and the filtrate was added
with acetic acid (CH3COOH) and then added with 1 Litre water.
In a test tube, 1 ml of 2% albumin solution and 1 ml of Hopkins cole reagent was mixed. The test tube
was inclined and 1ml of pure conc. H2SO4 was added on the side of the tube resulting to a formation of
a violet ring.
When protein is mixed with glyoxylic acid (CHOCOOH) is treated with sulfuric acid, a violet ring is
produced at the point of contact of the two solutions. This is due to the presence of an idole nucleus in
the tryptophan component. The tryptophan condenses with the aldehyde to form the colored
compound.]
Heller’s ring test
The test is used clinically to detect the presence of albumin in urine.
In a test tube a 5 ml of con. Nitric acid was inclined in the tube and allowed an albumin solution to
slowly flow down the side of the tube.
The interface between two liquids formed a white ring or resulted a white precipitate which indicates
that the Heller’s ring test is positive.
The nitric acid represents as a lower layer beneath the solution of protein, and then a white precipitate
of coagulated proteins appears at the junction of the two layers.
Reduced sulfur test:
Loosely combined sulfur after boiling with sodium hydroxide forms sodium sulfide. A positive test is
given by amino acids which contain the reduced sulfur group.
In a test tube, a 2 ml of 2% albumin solution was added to a 2 ml of 40% NaOH and 10 drops of 2% lead
acetate solution. It was then headed for about 2 minutes or up until there was an obtained change.
Amino acids that are heated with NaOH, it splits up the sulfur to form Na2S. The amino acid was added
by NaOH, and then was treated with lead acetate, it produced a black precipitate due to the formation
of lead sulfide.
Adamkiewicz reaction
In a test tube, 3 drops of 2% albumin solution was added to a 5 ml Glacial acetic acid. It was mixed well.
After mixing, conc. Sulfuric Acid was added down to the side of the tube.
The solution has a vinegar-like odor of glacial acetic acid and albumin solution which separates sulfuric
acid. The formation of a violet ring which indicates that the Adamkiewicz reaction was positive.

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Color Reactions of Proteins

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COLOR REACTIONS OF PROTEINS

Heller’s ring test

The test is used clinically to detect the presence of albumin in urine.

Reduced sulfur test:

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