COLOR REACTION OF PROTEINS

COLOR REACTION OF PROTEINS

Proteins are the major constituents in every living cell. They have a
variety of functions. They catalyze biochemical reactions, regulate the
activity of various organs in the body, counteract the adverse effects of
antigens, transport molecular oxygen, and serve as structural materials of
the muscle, skin and hair.
Chemically, proteins are macromolecule that contain alpha amino
acids as the building blocks. These amino acid are joined together by the
peptide bond.
Objectives: To determine the chemical properties through color reaction
tests.
Reagents: 1% of the following solutions, casein, peptone, albumin, CuSO4
and phenol, 10% NaOH, Millon’s reagent, conc. HNO3 and NH4OH, conc.
H2SO4, and 5% lead acetate.
BIURET TEST

Purpose: This test is utilized to identify if peptide bonds are present in

proteins.

Principle: The biuret reagent reacts with peptide bonds in proteins to form a
blue to violet complex known as “biuret complex”. Two peptide bonds are
required for the formation of this complex.
Reagents used: 10 % NaOH and 1% CuSO4

Test Solution: 1 % albumin, 1% casein, 1 % peptone, urea w/ distilled water

RESULTS:
POSITIVE – Violet
(presence of peptide bond)
NEGATIVE – Pale blue
(absence of peptide bond)
RESULTS
SOLUTION OBSERVATION INTERPRETATION

1% albumin  Violet Presence of Protein

1% casein Violet Presence of Protein

1% peptone Pink Low concentration of

peptide bonds

SOLUTION OBSERVATION INTERPRETATION

Urea with Violet Peptide bonds are
distilled present
water
MILLON’S REACTION
Purpose: To detect amino acid containing phenol group (hydroxyl
group attached to benzene ring) i.e. Tyrosine
Principle: Compounds containing hydroxybenzene radical react with
Millon’s reagent to form red complexes. The only amino acid having
hydroxybenzene ring is tyrosine. Thus, this test is specific for the
amino acid tyrosine and the protein containing this amino acid.
Tyrosine when reacted with acidified mercuric sulphate solution gives
yellow precipitate of mercury-amino acid complex. On addition of
sodium nitrate solution and heating, the yellow complex of mercury-
amino acid complex converts to mercury phenolate which is in red
color.
Reagents: Millon’s reagent (Acidified mercuric sulphate)
and 1% phenol
Results: Positive Millon’s test: Brick red color (Tyrosine and phenol solution)

Negative Millon’s test: no red color

**all phenol give positive Millon’s test

 2.

RESULTS
SOLUTION w/ OBSERVATION INTERPRETATION
millon’s reagent
1 % Albumin Brick red (Tyrosine and phenol
solution)
1 % Gelatin No red color (absence of phenol
solution)
1 % Casein Brick red (Tyrosine and phenol
solution)
 SOLUTION OBSERVATION INTERPRETATION
w/phenol
1 % Albumin Brick red (Tyrosine and
phenol solution)
1 % Gelatin Brick red (Tyrosine and
 
phenol solution)
1 % Casein Brick red (Tyrosine and
 
phenol solution)

SOLUTION OBSERVATION INTERPRETATION

20 drops of 1% Brick Red all phenol give

phenol positive Millon’s
test
XANTHOPROTEIC REACTION
Purpose: This test is performed to identify the amino acid having
benzene ring in them like tyrosine, tryptophan, and phenylalanine.

Principle: Treating aromatic amino acids with the concentrated

nitric acid leads to the nitration of the aromatic ring and formation of
yellow nitro-products (nitro derivatives). When the strong basic
solution is added the color of obtained products turns darker (from
yellow to orange). The positive result of xanthoproteic reaction gives:
tyrosine, tryptophan and phenylalanine (only after extended heating
time). It is difficult to nitrate phenylalanine under normal conditions
so it does not respond to this test without extended heating.

Reagents: Conc. HNO3 (Nitric acid), Conc. NH4OH (Ammonium

Hydroxide) and 1% phenol solution
Results: Positive : Color changes to yellow and orange
indicating the presence of aromatic amino acids
Negative – color does not change
 RESULTS
SOLUTION w/ OBSERVATON INTERPRETATION
HNO3 and
NH4OH
1 % Albumin Orange (Color changes to orange
  indicating the presence of
aromatic amino acids)
1 % Gelatin No color change (Color does not change to yellow
  indicating the absence of
aromatic amino acids)
1 % Casein Orange (Color changes to orange
  indicating the presence of
aromatic amino acids)
 SOLUTION OBSERVATION INTERPRETATION

10 drops of 1% ORANGE (Color changes to

phenol orange indicating
the presence of
aromatic amino
acids)
TEST FOR SULFUR
Purpose: to detect amino acid containing sulfhydryl
group (-SH) i.e. cysteine

Principle: Proteins containing sulfur (like: cysteine)

give a black deposit of lead sulfide (PbS) when heated
with lead acetate in alkaline medium. When cysteine is
heated with strong alkali like NaOH, some of the
sulphur is converted to sodium sulphide (Na2S) which
can be detected by precipitation as lead sulphide (PbS)
from alkaline solution.

Reagents: 5% Lead acetate and 10% NaOH

Results: Positive – Black precipitate
Negative- no black ppt
Sulfur-containing protein ---->
NaOH----> Na2S- ----+Pb2----> PbS
 RESULTS
SOLUTION OBSERVATION INTERPRETATION
Albumin Black ppt Presence of sulfur
Solid Gelatin No black ppt Presence of
Methionine
Peptone Black ppt Presence of sulfur
 HOPKIN’S-COLE REACTION
Purpose: It is also known as the glyoxylic acid reaction.
This test used for detecting the presence of tryptophan
in proteins.
Principle: The indole group of tryptophan reacts with
glyoxylic acid in the presence of conc. H2S04 to give a
purple color. Glyoxylic acid is prepared by reducing
oxalic acid with magnesium powder or sodium
amalgam. Glacial acetic acid which has been exposed
to the sunlight also contains glyoxylic acid and can
thus be used for this test.
Reagents: Hopkins Cole Reagent and conc. H2SO4
Results: Positive – Violet ring formed between the two
layers due to presence of tryptophan
Negative – No appearance of violet ring
RESULTS
SOLUTION OBSERVATION INTERPRETATION
1 % Albumin Violet ring (Violet ring appears between
the two layers due to
presence of tryptophan)
1 % Gelatin No Violet ring (No appearance of a violet
  ring between the two layers
due to absence of
tryptophan)
1 % Casein Violet ring (Violet ring appears between
  the two layers due to
presence of tryptophan)
 NINHYDRIN REACTION
Purpose: Used to determine the presence of free amino acids in a
solution
Principle: Reaction of amino acids with ninhydrin leads to their
decarboxylation, deamination (formation of CO2 and ammonia)
and formation of aldehyde which has one carbon atom less in its
structure. The ninhydrin undergoes reduction to form
hydrindantin. The reduced ninhydrin condenses with ammonia
and non- reduced ninhydrin molecule which leads to the
formation of the blue-purple condensation product called
Ruhemann’s purple.
Reagents: Freshly prepared ninhydrin solution
Results: Positive test: Blue-purple reaction products positively
identify free amino groups on amino acids and
proteins.
Negative test: No change (absence of amino acids and
proteins)
RESULTS
SOLUTION w/ OBSERVATION INTERPRETATI
ninhydrin solution ON
Albumin solution Blue-purple Presence of
amino acids
SOLUTION OBSERVATION INTERPRETATION
Ammonia Water Pale Purple NO presence of
Amino Acids
0.2% Urea Pale Purple NO presence of
amino acids
0.2% Glycine Blue - Purple Presence of Amino
acids
GUIDE QUESTIONS:
1. Why do all proteins give a positive in
Biuret test?
It gives positive result to any compound
containing two carbonyl groups attached to
a nitrogen or carbon atom. Thus, it may
not be completely protein-specific.
2. Why does nitric acid strain the skin
with a yellow color?
Proteins and amino acids that contain
phenyl rings form a yellow colored
compound when treated with
concentrated nitric acid. When nitric acid
comes in contact with the skin, it reacts
with the protein, keratin to produce
yellow color nitrated products called
xanthoproteic acid.
3. Will methionine give a positive sulfur test? Support
your explanation with the chemical structure of
methionine.
No, Methionine does not give lead sulfide test
because sulfur in methionine does not split in
presence of alkali. Sulphur in methionine is inert, it
does not readily react with NaOH to form sodium
sulfide which would form black/brown ppt. with lead
acetate. Getting this ppt. is the positive response in
sulfur test for amino acids.
4. What grouping in amino acid or
proteins is responsible for the ninhydrin
reaction?
Ninhydrin is used in many
bioanalytical techniques particularly for
amino acid analysis method. Ninhydrin
reacts with the α-amino group of
primary amino acids producing
'Ruhemann's purple'.
5. What color will be produced by the
proline with ninhydrin? Why?
Proline, a secondary amine, gives a
yellow-orange product. Proline has
aliphatic side chains with a distinctive
cyclic structure. Proline does not give the
ninhydrin reaction as this reagent requires
free alpha amino group (-NH2) but proline
has an imino group (-NH). For the amino
acids which have a free –NH2 group,
ninhydrin test is positive but is negative
for proline because it only has –NH group.
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