COLOR REACTIONS

OF INTACT PROTEIN

Presented by: Group 1

PROTEINS

Proteins are the most characteristic

chemical components found in the living
cell.
Proteins are considered to be the building
blocks of life.
They have high molecular weights, and
each protein molecule is composed of 20
different kinds of amino acids linked to
each other in large numbers, and in
varying sequences and configuration.

POST LAB BIURET TEST

a) What type of chemical grouping is present in
all proteins?
- These are amino acids which contain the
amino group, NH2, and the acid group, COOH.

b) How many of these chemical groupings must

be present in a molecule to give a positive biuret
test?
- All of these chemical groupings must be present
to give a positive biuret test.

POST LAB BIURET TEST

c) Give the principle involved and the chemical
structure responsible for the positive biuret test
The biuret reaction is used to assess
theconcentration or presence of proteins because
peptide bonds occur with the same frequency per
amino acid in the peptide.
The biuret reaction is based on the reduction of
Cu2+ to Cu+ which then complexes with the nitrogen
atoms on the peptide bonds. This reaction is carried
out at high pH and gives a violet color. It is the amide
bond (common to all proteins) which is responsible
for the color.

POST LAB NINHYDRIN TEST

a) Is the shade of color produced in a positive ninhydrin
test always the same? Give reason to your answer
- No. Usually in the Ninhydrin test, Ninhydrin
degrades amino acids into aldehydes, ammonia, and
CO2the net result is hydrindantin. Ninhydrin then
condenses with ammonia and hydrindantin to produce a
blueish-purple pigment which is the typical positive
result.
However,
there
is
an
amino
acid
thatgivesayellowresult, which is proline. Proline has a
ring structure (secondary amino acid).
The blueish-purple result is usually associated with
primary amino acids. In these amino acids, the N is free
to react with ninhydrin.
o

POST LAB NINHYDRIN TEST

b) Can you distinguish proteins and other amino
compounds by the ninhydrin test? Explain briefly
- Yes since the ninhydrin test is a test to detect
proteins and amino acids. Proteins are made up of
hundreds of smaller units of amino acids
Ninhydrin is an oxidating agentwhich leads to the
oxidative deamination of alpha-amino groups. It is
very important for the detection and the quantitative
analysis of amino acids. Ninhyndrin test is used to
detect ammonia (deep blue or purple color) and
secondary amines ( yellow) this is a proof of free amino
acids in proteins.
o

POST LAB NINHYDRIN TEST

c) Would the ninhydrin test be useful and reliable
for showing the presence or the absence of
proteins?
- Yes, because the Ninhydrin Test is a test for
amino acids and proteins with a free -NH2group.

POST LAB XANTHOPROTEIC TEST

o

a) Do all amino acids having an aromatic ring give a

positive xanthoproteic test under identical
conditions? Give reason for your answer

- Yes, all amino acids with aromatic rings give a

positive reaction which usually involves a yellow
coloration due to the nitrated aromatic amino acid.
It is due to the nucleophilic content of an aromatic
ring that gave the amino acid a property to be nitrated.
Therefore we can say that all aromatic ring amino acids
give a positive reaction

POST LAB XANTHOPROTEIC TEST

b) Do most proteins give a positive xanthoproteic test?
-Yes since proteins are made up of smaller units of
amino acids. Thetest gives a positiveresult in
thoseproteinswhich contains amino acids carrying
aromatic groups, especially in the presence of tyrosine.
Since proteins contains tyrosine and tryptophan it will
give a positive resultThe xanthoproteic test is based on a
nitration reaction which is used to identify the presence
of an activated benzene ring. The amino acids tyrosine
and tryptophan contain activated benzene rings which
can readily undergo nitration and will change the
solution color to yellow in the xanthoproteic test.
o

POST LAB MILLONS TEST

a) What chemical structure is necessary for a positive
Millons test?
-Millons test is given by any compund containing a
phenoloc hydroxy group. Any protein containing
tyrosine will give a positive test of a pink-dark red color.
The hopkins-cole test is used to determine the presence
of the amino acid tryptophan.
b) Propose a probable mechanism for the above
reaction

POST LAB HOPKINS-COLE TEST

a) What chemical grouping is responsible for a positive
Hopkins Cole test?
- Tryptophan. It is an a-amino acidthat is used in
thebiosynthesisofproteins. It contains an -amino
group, an -carboxylic acidgroup and a side
chainindole, classifying it as a non-polar,aromatic amino
acid.
b) What is the role of concentrated H 2SO4 in the above
test?
-The indole group of tryptophan will react with
glyoxylic acid in the presence of concentrated H2SO4 to
give a purple color.Role of H2SO4 is to hydrolyzed the
tryptophan.

POST LAB SAKAGUCHI TEST

a) Why should there be an order of addition of the
reagents in the Sakaguchis test?
-The Sakaguchi test is performed with what is
known as the Sakaguchi reagent, a combination of 1naphthol and sodium hypobromite. When the reactant
is added, the guanidine group in arginine reacts with it
to form a reddish color.
Sakaguchi test are fairly dangerous.This test is
extremely sensitive and especially if there will be an in
order addition of reagent.
b) How could the colored product be stabilized for
further studies?

POST LAB PB (AC) 2TESTS

a) What element in the amino acid is detected to be
present in this tests?
- Element Arginine. It is an -amino acid that is
used in the biosynthesis of proteins. It contains an amino group, an -carboxylic acid group, and a side
chain of a 3-carbon aliphatic straight chain capped by
a complex guanidinium, classifying it as a charged
aliphatic amino acid.

COLOR REACTIONS OF INTACT

PROTEIN
Tests
A. Biuret
B. Ninhydrin
C. Xanthoproteic
D .Millons
E. Hopkins-Cole
F. Sakaguchi
G. Pb (Ac) 2

Casien /
Skimmed milk

Gluten/Flour

Bean Protein

Blue

Blue

Light blue

Yellowish

Clear

Blue/visible

Light yellow

Slight yellow/

blue
Yellow

Clear

clear
Clear

Pinkish/red

suspension

suspension

suspension

Clear

Clear

Clear

suspension

suspension

suspension

BIURET TEST

NINHYDRIN TEST

XANTHOPROTEIC TEST

MILLONS TEST

HOPKINS-COLE TEST

SAKAGUCHI TEST

PB (AC) 2 TEST