ISOLATION AND HYDROLYSIS OF CASEIN FROM NON-FAT MILK AND IDENTIFICATION

OF AMINO ACIDS THROUGH PAPER CHROMATOGRAPHY

Gabinete, A., Garcia, J. Gomez, L., Gonzales, E., Juntarciego, J.
2A-PH, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Casein is the most abundant protein in milk. It is an excellent source of all the essential amino acids
and is a slow-digesting protein resulting in a slow but steady release of amino acids into circulation. In
this experiment, casein is isolated with the aid of 10% acetic acid and enzymatic hydrolysed with the
presence of proteases since they cut peptide bonds of proteins at specific amino acid residues. Since milk
is a source of complete protein, all 20 alpha amino acids are found in casein. This is justified by testing
the intact protein with qualitative color reactions giving positive results to ten different tests. Although
several amino acids are still detected in the enzyme hydrolysate, not all 20 alpha amino acids are found
since the hydrolysis caused breakage of peptide bonds between two amino acids in the composition of
casein. Standard serine amino acid and the enzyme hydrolysate obtained equal Rf values.

I.

INTRODUCTION

Proteins are large biomolecules consisting of

one or more long chains of amino acid residues.
Proteins are essential nutrients for the human
body because they are one of the building blocks
of body tissue and serve as a fuel source. Protein
is found throughout the body and makes up
enzymes and haemoglobin that carries oxygen in
the blood. According to The Institute of
Medicine, an adult should receive a minimum of
8 grams of protein for every 20 pounds of body
weight. [1] Phenylalanine, threonine, valine,
tryptophan, isoleucine, methionine, histidine,
lysine, and leucine are proteins which are not
synthesized by the body to supply the normal
requirements for protein biosynthesis and
categorized as essential proteins.[2]
A complete protein is a source of protein
that contains an adequate proportion of all nine
of the essential amino acids. Meat, products
from milk, eggs and fish are good sources of
complete protein. The primary group of milk
proteins is casein. Most of the milk proteins,
including casein, alpha-lactalbumin and betalactoglobulin, are derived from free amino acids
in the circulation. Casein is a family of

phosphoproteins that self-assemble into small

particles which then further assemble into units
known micelles. [3] Casein contains abundant
calcium and phosphorus. The casein of cows
milk contains 80% of the total milk protein.
The main objective of this experiment is to
isolate casein from skimmed milk by isoelectric
precipitation. The experiment also aims to
perform acid, alkaline, and enzymatic hydrolysis
on the isolated proteins, enumerate the
advantages and disadvantages of each type of
hydrolysis, and to analyse chemical groups
responsible for color reactions and explain the
principles behind in each test.
II.

MATERIALS AND METHODS

Four different procedures were done to

isolate the protein, to hydrolyse the intact
protein, to test for color reactions, and to
identify amino acids separately.
Casein Isolation
105 mL of Nestle non-fat milk (high in calcium)
was measured and placed in a beaker, then
heated to about 40C using the hot plate. With

the aid of the thermometer, the mixture was kept

from heating exceeding 40C. 10% acetic acid
was added dropwise and was gently stirred when
the mixture has reached 40C. Acetic acid was
continuously added until precipitates formed.
The use of pH meter to measure pH change to
4.6 was modified due to absence of a pH meter.
The congealed casein was filtered by gravity
filtration (Figure 1). The isolated protein was
divided into four equal parts for hydrolysis and
chromatography.
Enzymatic Hydrolysis of Intact Protein
of the divided isolated protein was assigned
for the enzymatic hydrolysis of intact protein. 1
g of the isolated protein and 100 mL distilled
water was prepared to create a protein mixture.
50 mL of the protein mixture was added to 0.050
g of protease, then, 10 mL of 0.1 M phosphate
buffer having a pH of 7.5 was added as well.
The tube was kept inside the locker overnight
for digestion to occur. On the next meeting, the
hydrolysed mixture was used for the next
procedure on qualitative analysis.
Qualitative Color Reactions
0.5 g of casein in 1 mL distilled water and 0.5
mL of the enzymatic hydrolyzed sample were
used for each test. Each of the test tubes was
treated with different reagents for different tests.
In Biuret Test, 20 drops of 2.5 M NaOH and 3
drops of 0.1 M CuSO4 solution were added in to
the samples. The tubes were shook to note the
color of the solution.
In Ninhydrin Test, 10 drops of 0.1% ninhydrin
solution was diluted into the samples, then the
tubes were heated in a boiling water bath.
In Xanthoproteic Test, 10 drops of concentrated
HNO3 and 10 drops of concentrated NaOH were
slowly added to the diluted samples.
tyrosine, histidine, glycine, and aline were
applied five times separately on different points.
The three hydrolyzed samples were applied ten
times. Capillary tubes were used in application
and samples were dried in between applications
to prevent overlapping of plotted samples. The

5 drops of Millons reagents were added to the

diluted samples for the Millon Test. In HopkinsCole Test, 20 drops of Hopkins-Cole reagent
was mixed into the samples. The test tubes were
inclined for the slow addition to the side of 20
drops concentrated H2SO4.
For Sakaguchi Test, 10 drops of 10% NaOH and
10 drops of 0.02% naphthol solution were added
to the samples. After the samples were allowed
to stand for 3 minutes, 3 drops of 2% NaOBr
was added.
In Nitroprusside Test, 0.5 mL of 3 M NaOH and
0.25 mL of 2% nitroprusside solution were
added to the samples.
In Fohls test, 5 drops of 30% NaOH and 2
drops of 5% Pb(CH3COO)2 were added to the
samples. The tubes were then placed in a boiling
water bath.
To test for amides, 1 mL of 20% NaOH was
added to the sample. The tubes were placed in a
boiling water bath. During heating, evolution of
gas was tested by placing a moistened red litmus
paper.
In Pauly Test, the diazo reagent was prepared by
mixing 5 drops of 1% sulfanilic acid with 3
drops of 5% NaNO2 solution; the diazo reagent
and 5 drops of 10% Na2CO3 were added to the
sample.
Separation and Identification of Amino Acids
Paper chromatography was prepared by drawing
the origin in a pencil line across the filter paper
with 1.5-cm margin from the bottom. 13
equidistant points were marked on the line for
the spotting of the amino acids and three
hydrolysate samples. 2% w/v of tryptophan,
arginine, proline, cysteine, serine, aspartic acid,

paper was placed inside a pre-equilibrated

chamber containing the solvent 1-Butanol:acetic
acid:water (4:1:5). The solvent was allowed to
ascend undisturbed. The paper was removed
from the chamber when the solvent front
reached approximately 0.5 cm from the top edge

of the paper. The solvent front was marked with

a pencil line. The chromatogram was air-dried
and sprayed lightly with 1% ninhydrin reagent,
then placed inside an oven for 3 minutes. After
encircling all the spots, the Rf values were
computed (Figure 3). Rf value is computed
using this formula:

Rf =

disance traveled by compound

distance traveled by solvent

III.

RESULTS AND DISCUSSION

Different tests, such as solubility test, stability

test for alkali, and specific reactions on
individual amino acids, are can be performed for
the qualitative analysis of amino acid isolated
and hydrolysed. Figure 4 shows the results
obtained from the qualitative analysis of color
reactions. The following tests enumerated below
detect the presence of amino acids and
distinguish the amino acid present in the protein
from one another. [4]
Biuret Test
Both the intact casein and enzyme hydrolysate
produced purple solutions. The biuret test
identifies the presence of proteins in solution
with a deep violet color caused by cupric ions in
alkaline medium. The reaction is so named
because biuret, a compounded first obtained in
1848 also gives reddish0violet color with
prolonged heating of urea.[5] This needs a
minimum of two peptide bonds so individual
amino acids and dipeptides will not answer this
test.
Ninhydrin Test
Only the enzyme hydrolsate produced a blue
solution while the intact casein maintained a
clear solution. When an amino group is attached
to the alpha carbon on the amino acids carbon
chain, the amino group of nitrogen atom
becomes a blue-purple product. Amino acids
which have secondary amino group attachment
also react with ninhydrin though results to
yellow product.
Xanthoproteic Test

The intact casein did not change in color while

the enzyme hydrolysate produced an orange
solution.
Aromatic amino acids, such as
phenylalanine, tyrosine, and tryptophan, respond
to this test. With concentrated nitric acid, the
aromatic phenyl ring is nitrated to result into a
yellow colored nitro-derivatives. The color
changes to orange due to the ionization of the
phenolic group at alkaline pH.
Millons Test
In the experiment, neither one of the samples
prepared produced the visible positive result.
This test is specific for tyrosine. The phenol
group of tyrosine is nitrated by nitric acid in the
test solution. Then the nitrated tyrosine
complexes mercury (I) and mercury (II) ions in
the solution to form a red solution.
Color Reaction
Biuret
Ninhydrin
Xanthoproteic
Millons
Hopkins-Cole
Sakaguchi
Nitroprusside
Fohls
Test for Amides
Pauly

Intact Protein
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Enzymatic
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---orange----

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Aaaa aaaaa
Aaaaaaaaami

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Aaaa aaaaa
Aaaaaaaaaaaa

Figure 1 Reactions of intact casein and enzyme

hydrolysate

Hopkins-Cole Test
Both intact casein and enzymatic hydrolysate
produced a purple interface which is the visible
positive result of this test. Hopkins-Cole test is
specific for tryptophan, the only amino acid
containing an indole group. The indole ring
reacts with glyoxylic acid in the presence of
strong acid to form a violet cyclic product.
Sakaguchi Test
Neither one of the samples produced the desired
red solution. This test detects the presence of
free or intact arginine. Under alkaline condition,
alpha naphthol reacts with a mono-substituted
guanidine compound which upon treatment with
hypobromite produces a red color.

Nitroprusside Test
Neither one of the samples produced a visible
positive result. With sodium nitroprusside in
ammoniacal solution, proteins with free SH
groups give reddish color. This test is specific
for cysteine, the only amino acid containg a
sulfhydryl group.

5.7% tyrosine, 5.5% isoleucine, 4.5%

phenylalanine, 4.4% threonine, 3.7% arginine,
2.8% histidine, 2.7% alanine, 2.5% methionine,
2.4% glycine, 1.1% tryptophan, and 0.3%
cysteine.
0.5
0.4
0.3
0.2
0.1
0

Fohls Test
The intact casein gave a yellow solution while
the enzymatic hydrolysate produced a black
precipitate, which is the visible positive result
expected. Fohls test is a test for sulphur
containing amino acids like methionine. The
dark coloration of the samples tells us that there
is sulfur in the sample used.
Test for Amide
Although only the edge of the litmus paper
turned to blue in the evolution of gas, both the
intact casein and enzymatic hydrolysate showed
visible positive result. The test for amide is used
to detect R-groups of asparagine. The litmus
paper is expected to change into blue because of
basic hydrolysis.
Pauly Test
Both intact casein and enzymatic hydrolysate
produced red solution. This test confirms the
presence of histidine or tyrosine. Diazo-benzene
sulfonic acid reacts with imidazole group of
histidine to form a cherry-red colored diazotized
product under alkaline environment. On the
other hand, it will fom an orange red colored
product with phenol group of tyrosine.
From the data gathered in the experiment, we
can conclude that almost all of the 20 alpha
amino acids were detected in the color reaction
tests but most amino acids were present in the
intact protein compared to the enzymatic
hydrolysate since it was already hydrolysed in
the process. The undetected amino acids are still
present in the isolated protein from non-fat milk
but are already inactive. According to caseins
chemical formula, it contains 20.2% glutamic
acid, 10.2% proline, 8.3% leucine, 7.4% lysine,
6.5% valine, 6.4% aspartic acid, 5.7% serine,

Standards
Enzyme Hydrolysate
Acid Hydrolysate
Base Hydrolysate

Figure 2 Rf values of standard amino acids

The compositions of the stationary and mobile

phases define a specific chromatographic
method. The relative affinity of a substance for
each phase depends on properties such as
molecular weight, structure and shape of the
molecule, and the polarity of the molecule. As
the mobile phase rises on the paper, it spots
each amino acids and hydrolysed samples. (See
figure 5.) If an amino acid has a higher affinity
for the mobile phase than the stationary phase, it
will tned to travel with the solvent front while if
the amino acid has a higher affinity for the paper
than the solvent, it will tend to stick to the paper
and travel more slowly.[6] In figure 5, tryptophan
has the highest affinity to the mobile phase
while histidine has the highest affinity to the
paper. The affinities of these amino acids for the
mobile phase are correlated to the solubility of
the different amino acids in the solvent.
Comparing the Rf values obtained from the
standard amino acids and enzyme hydrolysate,
the enzyme hydrolysate got the same Rf value
with serine. This depicts the equivalent affinity
of the enzyme hydrolysate to the solvent and
aids us in concluding that there is an equal
amount of serine in the enzyme.
IV.
[1]

REFERENCES

What Should I Eat? Protein. Retrieved March

3,
2015
from
http://www.hsph.harvard.edu/nutritionsource/wh
at-should-you-eat/protein/

[2]

Di Pasquale, M. (1997). Amino Acids and

Proteins for the Athlete Nutrition in Exercise &
Sport. Florida, United Sates of America: CRC
Press

[4]

[3]

[5]

Patton, S. (2004). Milk: Its Remarkable

Contribution to Human Health and Well-Being.
New Jersey, United States of America:
Transaction Publishers

Sreekumari, S., Vaidyanathan, K. (2013).

Textbook in Biochemistry for Medical Students,
7th ed. Daryaganj, New Delhi: Jaypee Brothers
Medical Publishers Ltd
Rosenfeld, L. (1999). Four Centuries of
Clinical Chemistry. CRC Press
[6]

Sherma, J. & Zweig, G. (1971). Paper

Chromatography, vol. 2. New York, United
States of America: Academic Press, Inc.