CASEIN

PROTEIN ISOLATION AND CHARACTERIZATION OF ENZYMATIC

HYDROLYSATE BY PAPER CHROMATOGRAPHY AND QUALITATIVE COLOR
REACTIONS
2DPH Group 3
Constantino, Faye; Dagcuta, Arnoika; *De Alva, Mycaela; Decena, Marck Paulo;
Enriquez, Floen Michael

ABSTRACT
Casein is the main protein found in the milk of mammals including cows, goats,
and humans. Casein is the predominant phosphoprotein ( 1, S2, , k) that
accounts for nearly 80 percent of proteins in cow milk and cheese. Milk-clotting
proteases act on the soluble portion of the caseins, K-Casein, thus originating an
unstable micellar state that results in clot formation. It is recognized for its excellent
amino acid profile,slow digestion, and interesting peptides (casomorphins,
casokinins, casoxins, etc). In this experiment, Casein was isolated, hydrolyzed and
neutralized from non-fat powdered milk. Casein was first isolated by adding 10%
Acetic acid until if formed an amorphous mass. The isolated casein was further
hydrolyzed and neutralized for the characterization. A positive result from the
enzymatic hydrolysate was observed under Biuret, Ninhydrin, Xanthoproteic,
Hopkins-Cole, Nitropusside, Fohls, Test for amides and Pauly Tests. In the paper
chromatography, serine has the lowest Rf value with 0.07 along with Arginine with a
Rf value of 0.08. Tyrosine has the highest Rf value with 0.24.

I.
Introduction
qualitative and quantitative analysis.
Proteins can be considered as
The biuret test is used to detect the
polymers of amino acids. Amino acids
presence of peptide bonds while the
are linked by covalent peptide bond
Ninhydrin reaction is a typical test for
into linear chain, which is called
an -amino acid. Xanthoproteic test
peptide or polypeptide chain. The
detects side chains of aromatic amino
properties common to all amino acids
acidswhile the Millons and Hopkins-
are due to the relative special
Cole tests determine tyrosine and
arrangements of the carboxyl and
tryptophan residues, respectively. The
amino groups. The physical and
Nitroprusside test is used to find out if
chemical properties unique to each
sulfur-containing amino acids are
amino acid are the result of the
present; test for amides is used to
structure and chemical properties of
detect R-groups of asparagine and
the R group. Several amino acids
glutamine. Test for amides is used to
combine to form peptide bonds.
detect R-groups of asparagine and
Proteins contain polypeptide units
glutamine.
(several peptide units). When a
The objective of the experiment is
protein is hydrolyzed, it breaks down
to determine the amino acid
into smaller units (tri and dipeptides),
components of casein, which can be
eventually forming amino acids.
done
by
partition
paper
Specific reactions are used for the
chromatography, which is widely
purpose of identifying amino acids
employed for the separation of amino
and proteins in biological media, for
acids. The solvent migrates along a

strip of paper and carries amino acid

dissolved in it
II.
Methodology
a. Paper Chromatography
A 12cm x 15cm filter paper was
used
to
facilitate
paper
chromatography. A margin of 1.0 cm
was lined from the top and bottom to
the filter paper. Thirteen equidistant
points were marked to indicate where
the selected ten amino acids and the
three hydrolyzed proteins were to be
spotted. The ten amino acids were
spotted twice using a capillary tube
and the hydrolysates were spotted
five times. The filter paper was then
stapled to become a cylinder. A
1000mL beaker and a watch glass
were prepared to serve as the
developing chamber. The solvent
system used was a mixture of 1-
Butanol, acetic acid and water in 4:1:5
ratio and it was poured into the
beaker and left undisturbed inside the
developing chamber. Then the
cylindered filter paper was placed
inside the beaker, covered with watch
glass and allowed to ascend
uninterrupted for almost an hour.
The solvent front was marked
with a pencil and allowed to dry. The
chromatogram was sprayed with a 1%
Ninhydrin reagent and heated on top
of a hot plate for some minutes.
Purple and yellow spots were
observed and encircled.
Rf values were computed using
the following formula:
! =

Rf or retention factor is the

ration between the distance traveld by
the solute and the distance traveled by
the solvent. It is used to identify the
unknown by comparing its Rf value to
a Rf value of known compounds.

b. Qualitative Color Reactions

The reactions for side chains, -
amino and -carboxyl groups can be
used to characterized both free amino
acids and proteins. For each
colorimetric test, separate test tubes
of intact protein solution (0.5g of the
protein in the 1 mL distilled water)
and 0.5 mL of hydrolyzed sample were
used. The Biuret Test, Ninhydrin Test,
Xanthoproteic Test, Millons Test,
Hopkins-Cole Test, Sakaguchi test,
Nitropusside test, Fohls test, Test for
Amides, and Pauly Test were used to
characterize the free amino acids and
proteins.
Biuret Test: 20 drops of 2.5 M
NaOH was added to a test tube
containing the intact protein
and another 20 drops were
added to the test tube
containing the enzymatic
hydrolysate. Then to each test
tube, 2-3 drops of 0.1M CuSO
solution were added. Both test
tubes were shaken and the
color was noted.
Ninhydrin Test: In each test
tube,
6-10
drops
of
0.1%ninhydrin solution were
added into the intact protein
and enzymatic hydrolysate.
Both test tubes were then
heated in a boiling water bath.
Xanthoproteic Test: Ten drops
of concentrated HNO3 solution
was slowly added to the diluted
samples and were mixed.
Then,10 drops of concentrated
NaOH was added and the color
was noted.
Millons Test: To each of the
diluted samples, 5 drops of
Millons reagent were added

while noting the change in

color.
Hopkins-Cole Test: To the
diluted samples, 20 drops of
Hopkins-Cole reagent was
slowly added and mixed well.
The test tubes were then
inclined the test tube and 20
drops of concentrated H2SO4
was added along the side. The
change in color was then noted.
Sakaguchi Test: To each of the
diluted samples, 10 drops of
10%NaOH and 10 drops of
0.02%naphthol solution was
added and the test tubes were
then left untouched for 3
minutes.
Nitroprusside Test: 0.5 mL of
3M NaOH was added to the
diluted samples. Then, 0.25
mLof 2% of nitroprusside
solution was added.
Fohls test: Five drops of
30%NaOH and 2 drops of
5%(CH3COO)2Pb was added to
the diluted samples. Both test
tubes were then places in a
boiling water bath and the
change in color was observed.
Test for Amides: To each of the
diluted samples, 1 mL of
20%NaOH were added and
both test tubes were placed in
a boiling water bath. While in
the water bath, a red litmus
paper was held at the opening
of each test tube to test for the
evolution of gas.
Pauly
Test:
First,
the
diazoreagent was prepared by
mixing 3-5 drops of 1%
sulfanilic acid with 3 drops of
5% NaNO2 solution. Then, 5
drops of the sample and 3-5

drops of 10%Na2CO3 were

added to the diazoreagent. A
red coloration was noted.

III.
Results
a. Paper Chromatography
Table 1. Rf values of each amino acids
Enzymatic
Protein
Hydrolysate
Amino Acid
Distance Solvent
Standards
Rf
travelled front
Value
(cm)
(cm)
Tryptophan 1.89
9.0
0.21
Arginine
0.72
9.0
0.08
Proline
1.44
9.0
0.16
Cysteine
2.07
9.0
0.23
Serine
0.63
9.0
0.07
Aspartic
2.16
9.0
0.24
Acid
Tyrosine
3.78
9.0
0.42
Histidine
0.9
9.0
0.1
Glycine
1.8
9.0
0.2
Alanine
2.16
9.0
0.24

The table 1 shows the results of
the Separation and Identification of
amino
acids
by
Paper
Chromatography. Serine has the
lowest Rf value with 0.07. Followed
with Arginine with a Rf value of 0.08.
Tyrosine has the highest Rf value with
0.24.

Table 2. Qualitative Color Reactions
Color Reaction Enzymatic Protein
Hydrolysate
Biuret
+ (Purple solution)
Ninhydrin
+ (Purple solution)
Xanthoproteic + (Yellow solution)
Millons
- (White Turbid
solution)
Hopkins-Cole
+ (Pink interface)
Sakaguchi
- (Clear solution)
Nitropusside
+ (Yellow solution)
Fohls
+ (Clear solution)

Test for Amides + (Clear solution)

Pauly
+
(Red
orange
solution)

Table 2 shows the results of
qualitative color reactions of
enzymatic hydrolysate. A positive
result was observed under Biuret,
Ninhydrin, Xanthoproteic, Hopkins-
Cole, Nitropusside, Fohls, Test for
amides and Pauly Tests.

IV.
Discussion
a. Paper chromatography
Paper chromatography is the
separation and migration of the amino
acids based on its affinities to the
stationary and mobile phases.
Different factors affect the affinity of a
substance and these are the following:
polarity, pH, molecular weight,
structure, and shape of the molecule.
Based on the Table 1, amino acids
serine and arginine have the lowest Rf
value. Components with smaller Rf
value travel more slowly, a polar
compound, bond to the cellulose of the
paper more quickly and have a higher
affinity to the stationary phase. On the
other hand, tyrosine moved farthest
from the point of the origin.
Consequently, it has the greatest Rf
value. It means that tyrosine has the
greatest affinity toward the mobile
phase.
The Rf values of each component
indicated the polarities of the amino
acids. A higher Rf value denotes lower
polarity while a lower Rf value implies
that the component is polar.

b. Qualitative Color Reactions
The Biuret test is a test done
for the presence of peptide bonds. It is
a positive test for proteins but not for
amino acids. The evidence for a

peptide bond is the formation of a

violet-pink solution. The formation of
a violet-pink solution is due to when
the cupric ion, in a basic solution is
added to any polymer such as
proteins, which contains multiple
amide bonds. The enzymatic
hydrolysate shows positive result for
peptide bonds.

The Ninhydrin test is a test for
the presence of amino acid. The
positive result of amino acid is
detected by the yielding of a purple
solution. The enzymatic hydrolysate
showed negative results.
Xanthoprotheic test is used for
amino acids containing aromatic
groups that are derivatives of benzene
such as tyrosine and tryptophan.
These aromatic groups can undergo
reactions that are characteristics of
benzene and benzene derivatives. One
such reaction is the nitration of a
benzene ring with nitric acid. The
amino acids that have activated
benzene ring can readily undergo
nitration. This nitration reaction, in
the presence of activated benzene
ring, forms yellow product. The
enzymatic hydrolysate on the other
hand, showed some presence of
aromatic groups.

Millons test is specific for
the detection of tyrosine. Tyrosine is
the only amino acid that contains a
phenol group on which a hydroxyl
group is attached. A positive result
will yield a red precipitate. The
enzymatic hydrolysate showed a
negative result.
Hopkins-Cole test is specific for
the detection of the presence of
tryptophan. The color produced is due
to the formation of a compound from
the glyoxylic acid in the reagent and
the tryptophan in the protein. A

similar color is produced when

sulfuric acid is added to a protein
solution in the presence of a trace of
formaldehyde. The reaction is used as
a test for formaldehyde in milk. The
formation of a purple solution
indicates a positive result. The
enzymatic hydrolysate yielded a
positive result.

Sakaguchi test is used to test
the only amino acid,which contains a
guanidine group which is arginine.
Arginine gives a red color with
-naphthol, in the presence of an
oxidizing agent like Bromine solution.
The enzymatic hydrolysate showed
negative result for the presence of
arginine.

Nitroprusside Test is specific
for the detection of cystein, the only
amino acid containing sulfhydryl
group. This group reacts with
nitroprusside in the presence of
excess ammonia. Positive results show
a red complex. However, the
enzymatic
hydrolysate
showed
positive results, yielding a yellow
solution.

Fohls test is performed for
the determination of S- containing
amino acids. The solutions were
heated for the formation of sulfide. If
the test yielded a positive result, the
solution shows a red solution or a
further reaction of NA2S will lead to a
dark
brown
precipitate.
The
enzymatic hydrolysate yielded a
positive result.
Test for Amide is done for the
presence
of
Asparagine
and
Glutamine. When the red litmus paper
turned blue, it indicates a basic
component of the myoglobin and
enzymatic hydrolysate.

Pauly test is specific for
histidine and tyrosine wherein it deals

with the formation of azo dyes. Its

positive result is the formation of red
solution. The enzymatic hydrolysate
yielded a positive result.

V.
References
Crisosotomo, A. et.al Laboratory
Manual in General Biochemistry
Murray RF, Harper HW, Granner DK,
Mayes PA, Rodwell VW.
(2006).Harper'sIllustrated Biochemistr
y.

http://www.ncbi.nlm.nih.gov/
pmc/articles/PMC47762/