ISOLATION AND COLOR REACTIONS OF INTACT PROTEIN (CASEIN)

Ira A. Gica, Joseph Richard C. Guevarra, Aaron Terence D.C. Ibe,

Eden Venus L. Lagmay, Renze Jazmine B. Lucas, Anne Marinel K. Macalino
Group 4 2I Medical Technology
Biochemistry Laboratory
ABSTRACT
The experiment was performed to isolate the protein, casein, by isoelectric preparation and to analyze
chemical groups responsible for color reactions and explain the principle involved in each test. The
formation of a white solid precipitate determined the presence of casein in milk. The different color
reaction tests determined the presence of the following kinds of amino acids: alpha-amino acid, arginine,
aromatic amino acid, asparagine, glutamine, histidine, peptide bonds, sulfur containing amino acid,
tryptophan and tyrosine.

INTRODUCTION
Proteins are an essential constituent of all cells
and the most abundant organic molecules found
in living cells. Proteins can be classified by its
three-dimensional structure: fibrous and globular.
Fibrous proteins are long chains or sheets of
amino acids. Globular proteins are compact,
spheroidal-shaped amino acids.
They are naturally occurring polymers
composed of amino acids as its monomer which
are connected by peptide bonds. Peptide bonds
are amide bonds formed between the carboxylic
acid group and the carboxylic group of two
different amino acids.
The following are considered in the isolation of
an intact protein from its source: its 3D structure
(fibrous or globular), the interactions that keep
its native conformation functional (electrostatic,
covalent, hydrophobic, H-bonding, van der
Waals), its acid-base property and its solubility in
different solvents. [1]
Caseins, lactalbumins, and lactoglobulins are
globular proteins found in milk. Casein is a
phosphoprotein which has phosphate groups
attached to the hydroxyl groups of some of the
amino acids side chains. It exists in milk as the
calcium salt, calcium caseinate.
A weak acid, acetic acid, was added to milk to
isolate casein.
Certain functional groups in amino acids and
proteins react to produce characteristically
colored products. The color intensity of the
product formed by a particular group varies
among proteins in proportion to the number of
reacting functional groups or free groups present
and their accessibility to the reagent. The
following color reactions were performed to
characterize the protein and determine the
presence of a certain amino acid:

A. Biuret test
This test was performed to detect the
presence of peptide bonds. It should
produce a violet solution.
B. Ninhydrin test
This test was performed to detect the
presence of alpha-amino acid. Amino acids
contain free amino group and free
carboxylic group that react together with
Ninhydrin to produce a blue-violet
solution.
C. Xanthoproteic test
This test was performed to detect the
presence of the side chains of aromatic
amino acids which produces a yellow
solution. Xanthroproteic comes from a
Greek word that means yellow thus the
name for the test.
D. Millons test
This test was performed to detect the
presence of tyrosine residues. Red
precipitate formation or red solution
indicates a positive result.
E. Hopkins-Cole test
This test was performed to detect the
presence of tryptophan and produces a
violet solution.
F. Sakaguchi test
This test was performed to detect the
presence of arginine. The sample to be
tested was treated with alpha-naphthol
and sodium hypochlorite. A positive result
produces a reddish wine color.
G. Nitroprusside test
This test was performed to detect the
presence of cysteine, the only amino acid
with a sulfhydryl group (-SH). A red
solution should be obtained.

H. Fohls test
This test was performed to detect the
presence of sulfur containing amino acid.
A black or brown solution notes a positive
result.
I. Test for Amides
This test was performed to detect the
presence of asparagine and glutamine. A
yellow solution should be obtained.
J. Pauly test
This test was performed to detect the
presence of tyrosine or histidine.

C. Xanthoproteic test
Ten drops of concentrated HNO3 was
slowly added into the diluted sample
then mixed. The color of the solution
was noted. Ten drops of concentrated
NaOH was then slowly added and
mixed. Color of the solution was again
noted.

D. Millons test
Five drops of Millons reagent was
added into the diluted sample and its
change in color was noted.

E. Hopkins-Cole test
EXPERIMENTAL
A. Compounds tested (or Samples used)
Powdered non-fat milk

B. Procedure
1. Isolation of Protein
In order to isolate casein from milk, the
latters pH should be decreased at a point
where casein is insoluble, which is at pH
4.6. To adjust the pH, 10% acetic acid
was added drop wise to the solution while
being heated on a 40 degree Celsius
temperature. When the desired pH is
reached, curds formed and the milk
became almost opaque. This shows that
casein is being separated from it.
Casein was filtered by gravity filtration.
The decantate was set aside and the
casein residue was dried and its % weight
isolated was calculated.

2. Qualitative Color Reactions

For each test, test tubes were prepared
with an intact protein solution (0.5 g of
the protein in 1 mL distilled water) and
0.5 mL of the hydrolyzed sample.

A. Biuret test
Twenty drops of 2.5 M NaOH and two
to three drops of 0.1 M CuSO 4 was
added to the sample then mixed well.
Color of the solution was noted.

B. Ninhydrin test
Six to ten drops of 0.1% ninhydrin
solution was added into the diluted
sample then heated in a boiling water
bath. The appearance of a blue-violet
coloration was noted.

Twenty drops of Hopkins-Cole reagent

was slowly added into the sample and
mixed well. While the test tube was
inclined, twenty drops of concentrated
H2SO4 was slowly added along the
side. The mixture was not mixed and
the color at the interface was noted.

F. Sakaguchi test
Ten drops of 10% NaOH and ten drops
of 0.02% naphthol solution was added
into the sample and mixed well. After
three minutes, three drops of 2%
NaOBr was added into the solution and
mixed well. Color produced was noted.

G. Nitroprusside test
0.5 mL of 3 M NaOH was added to 0.5
mL of the sample. 0.25 mL of 2%
nitroprusside solution was added into
the solution and the formation of a red
solution was noted.

H. Fohls test
Five drops of 30% NaOH and two
drops of 5% (CH3COO)2Pb was added
into the sample. The tube was placed
in a boiling water bath and the
appearance of dark black or brown
sediment was noted.

I. Test for Amides

One mL of 20% NaOH was added to
ten drops of the sample. The tube was
placed in a boiling water bath and
evolution of gas was tested during
heating by placing a moistened red
litmus paper over the mouth of the
tube. The result was noted.

J. Pauly test
The diazo reagent was prepared by
mixing three to five drops of 1%
sufanilic acid with three drops 5%
NaNO2 solution. Five drops of the
sample and three to five drops of 10%
Na2CO3 was added to the diazo
reagent. The appearance of a red
coloration was noted.

RESULTS & DISCUSSION

The addition of acetic acid to milk formed a
precipitate, a white solid particle that confirms
the presence of casein in milk. The negativity of
casein was neutralized by the addition of the
10% acetic acid until it reached the isoelectric
point of casein which is 4.6 at which it is
insoluble, casein precipitated when the solution
reached the pH of 4.6.
Various color-producing reagents were used to
qualitatively detect the presence of certain
functional groups in casein.
Table 1. Color Reaction tests Results.
Color reaction test
A. Biuret test
B. Ninhydrin test
C.
D.
E.
F.
G.
H.

Xanthoproteic test
Millons test
Hopkins-Cole test
Sakaguchi test
Nitroprusside test
Fohls test

I.

Test for Amides

J.

Pauly test

Result
Violet solution
Reddish-brown precipitate
Blue-violet solution
Black precipitate
Yellow ppt.
Off-white or peach solution
Purple ring
Turbid
Yellow solution
Yellowish-brown solution
Brown sediments
Red to blue litmus paper
Orange solution
Red precipitate
Red-orange solution

The results obtained from the color reactions

were all positive which determined the presence
of the following amino acids: (A) Biuret test
confirmed the presence of peptide bonds, (B)
Ninhydrin test confirmed the presence of an
alpha-amino
acid,
(C)
Xanthoproteic
test
confirmed the presence of aromatic amino acid,
(D) Millons test confirmed the presence of
tyrosine, (E) Hopkins-Cole test confirmed the
presence of tryptophan, (F) Sakaguchi test
confirmed the presence of arginine, (G)
Nitroprusside test did not confirm the presence of
cysteine, (H) Fohls test confirmed the presence
of sulfur containing amino acid, (I) Test for Amide
confirmed the presence of asparagine and
glutamine and (J) Pauly test confirmed the
presence of tyrosine and histidine.
In conclusion, the formation of a white solid
precipitate determined the presence of casein in
milk and that it is composed of several amino
acids linked by a peptide bond. Through
performing the different color reaction tests, the
following kinds of amino acids were found to be
present: alpha-amino acid, arginine, aromatic
amino acid, asparagine, glutamine, histidine,
peptide bonds, sulfur containing amino acid,
tryptophan and tyrosine.

REFERENCES
[1]
Crisostomo, A.C., et. al. (2010)
Laboratory
Manual
in
General
Biochemistry
[2] Isolation of Casein from Milk
https://www.apsu.edu/sites/apsu.edu/file
s/chemistry/SP11_1021_ISOLATION_OF_
PROTEINS_FROM_MILK.pdf
[3] Qualitative Analysis of Amino Acids
http://vlab.amrita.edu/?
sub=3&brch=63&sim=1094&cnt=1