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Sharysse Pearl J. Acosta, Jan Andrei B. Almazan, Sean Christopher Almazan,

Erwin Jacobson T. Ang and Joshua Aldon S. Aparicio
Group 1 2A Medical Technology Biochemistry Laboratory

The group was assigned to isolate casein from skimmed milk using isoelectric precipitation. Ten percent acetic
acid was used to isolate casein at its isoelectric pH. The caseinate was then subjected to acid hydrolysis using 6M HCl.
After autoclaving of the acid hydrolyzed caseinate, it was used in performing different tests for qualitative color
reactions. The same tests were performed with the intact protein. After the qualitative color reactions, paper
chromatography was performed for the separation and identification of amino acid standards based on the polarities of
tryptophan, arginine, proline, cysteine, serine, aspartate, histidine, glycine, and alanine.

Protein isolation is a series of processes
intended to isolate one or a few proteins from a
characterization of function, structure and
interactions of the protein interest and also, to
determine their solubility and acid-base property.
Proteins can be separated depending on their
physiochemical properties. The most commonly
used methods are isoelectric precipitation, heat
In isolating casein from skimmed milk,
isoelectric precipitation was the method used.
Milk is a mixture of many types of proteins, most
of them present in very small amounts. Milk
proteins are classified into three main groups of
proteins on the basis of their widely different
behaviours and forms of existence and one of
these proteins is casein. Caseins are made up of
many hundreds of individual amino acids. Each
may have a positive or a negative charge,
depending on the pH of the system. At some pH
value, all the positive charges and all the
negative charges on the casein will be in balance,
so that the net charge on the protein will be
That pH value is known as the isoelectric
point (IEP) of the protein and is generally the pH
at which the protein is least soluble. For casein,
the IEP is approximately 4.6 and it is the pH
value at which acid casein is precipitated. In
skimmed milk, which has a pH of about 6.6, the
casein micelles have a net negative charge and
are quite stable. During the addition of acid to
milk, the negative charges on the outer surface
of the micelle are neutralized (the phosphate
groups are protonated), and the neutral protein

The same principle applies when milk is

fermented to curd. The lactic acid bacillus
produces lactic acid as the major metabolic endproduct of carbohydrate [lactose in milk]
fermentation. The lactic acid production lowers
the pH of milk to the IEP of casein. At this pH,
casein precipitates.
In acid hydrolysis, secondary, tertiary and
quaternary structures of the casein are lost in the
presence of 6M hydrochloric acid and water. This
process breaks covalent bonds of the amino acids
in the intact protein. Acid hydrolysis completely
hydrolyzes the peptide bonds and there is no
racemization. This was performed for the
preparation for qualitative color reaction tests
namely Biuret test (used to detect the presence
of peptide bonds), Ninhydrin test (typical test for
an alpha amino acid), Xanthoproteic test (detects
side chains of aromatic amino acids), Millons and
Hopkins-Cole tests (determine tyrosine and
tryptophan residues respectively), Nitroprusside
test and Fohls test (to find out if sulfurcontaining amino acids are present), Sakaguchi
test (for arginine), Pauly test (for histidine and
tyrosine) and lastly Amide test (used to detect Rgroups of asparagine and glutamine).
For the identification and separation of
amino acids, paper chromatography can be used.
It is a method of separating the components of a
mixture based on their differential affinity for two
phases, which are the stationary phase and
mobile phase. Polarity of proteins can also be
determined through this method.

A. Compounds tested (or samples used)
Casein, acid hyrolysate, tryptophan,
arginine, proline, cysteine, serine, aspartate,
histidine, glycine, alanine
B. Procedure
1. Isolation of Casein from Skimmed Milk

Twenty grams of powdered skimmed milk

was mixed with 50.0 mL of distilled water into a
100-mL beaker. The mixture was heated up to
40C then, 10% acetic acid was added dropwise
until curd was formed. The white curd-like
precipitate was filtered using a funnel and filter
paper. The filtrate was discarded and the residue
was divided into two portions, one portion was
refrigerated as an intact protein and the rest was
used for acid hydrolysis.

Figure 1. Checking the temperature of the

milk mixture to after heating

Figure 2. Casein precipitate

3. Acid Hydrolysis of Intact Protein

Five mL of 6M HCl was added to an
approximate amount of 0.5g isolated casein in a
hard glass test tube and was labeled and
autoclaved by the laboratory instructors. After
autoclaving, a dark brown-black solution was
formed which was then the acid hydrolyzed
casein. Ten mL distilled water was added to the
mixture and was neutralized with 1M NaOH.
4. Qualitative Color Reactions
Two sets of 10 test tubes were prepared
for the qualitative color reactions. The first set
contains the intact protein solution (0.5g of intact
casein in 1mL distilled H2O) and the second set of
test tube contains 0.5mL of the acid hydrolyzed
sample. The following tests were performed on
each of the two sets of sample:
Biuret test: 20 drops of 2.5M NaOH were
added and mixed well to the samples. Then, 2-3
drops of 0.1M CuSO4 was added.
Ninhydrin test: 6-10 drops of 0.1% ninhydrin
solution was placed into the sample then heated
in a boiling water bath.
Xanthoproteic test: 10 drops of concentrated
HNO3 was slowly added to the samples. After
which, 10 drops of concentrated NaOH was slowly
Millons test: 5 drops of Millons reagent was
added to the samples.
Hopkins-Cole test: 20 drops of Hopkins-Cole
reagent was mixed to the samples. Then each
test tube was inclined and 20 drops of
concentrated H2SO4 was slowly added.
Sakaguchi test: 10 drops of 10% NaOH and
10 drops of 0.02% napthol solution was added to
the samples. After 3 minutes, 3 drops of 2%
NaOBr was mixed to the samples.
Nitroprusside test: 0.5mL of 3M NaOH was
added to the samples. Then, 0.25mL of 2%
nitroprusside solution was added.
Fohls test: 5 drops of 30% NaOH and 2 drops
of 5% (CH3COO)2Pb was added to the samples
then the rest were placed in a boiling water bath.
Paulys test: diazo reagent was prepared by
mixing 3-5 drops 1% sulfanilic acid with 3 drops
5% NaNO2 solution. Five drops of the sample and
3-5 drops 10% Na2CO3 was added to the diazo
Test for amides: 1mL of 20% NaOH was
added to 10 drops of the sample. The samples
were then placed in a boiling water bath and red
litmus paper was placed and moistened over the
mouth of the tube.
3. Paper Chromatography
The solvent system was a mixture of 1Butanol: acetic acid: water in a 4:1:5 ratio

tryptophan, arginine, proline, cysteine, serine,

aspartic acid, tyrosine, histidine, glycine and
alanine were spotted on the paper chromatogram
using a capillary tube. After which, 1% ninhydrin
solution was sprayed to the chromatogram to
react with the protein identified then the paper
was place over the hot plate to dry.
A. Isolation of Proteins
Casein was isolated from skimmed milk
through the addition of acetic acid. As acetic acid
was added to the milk mixture at a controlled pH
level, a white curd-like precipitate was produced.
This process is called the isoelectric precipitation
where the protein has reached its isoelectric point
wherein its net charge is equal to zero.

Figure 3. Color reaction results with the

intact protein

B. Acid Hydrolysis of the Intact Casein

Acid hydrolysis was performed to breakdown
casein into peptides and amino acids. After
autoclaving of the acid hydrolysate, a dark
brown-black solution was formed.
C. Qualitative Color Reactions

Purple solution

Brown turbid


No color

coloration in
the solution


Very light
orange color

Light brown
turbid solution


White turbid

Light brown
turbid solution


Purple color of

Light brown
turbid solution


Red solution

Brown turbid


Yellow solution

brown turbid


Black brown

Brown solution
with black ppt.

Amide test

litmus paper

litmus paper


Red orange

Reddish brown

Table 1. Results of Color Reactions

Figure 4. Color reaction results with the acid

Each test was performed to confirm
different properties of casein and its acid
hydrolysate. The reactions of the intact proteins
and hydrolysates to the reagents of each test
depend on their characteristics.
Biuret test is a general test for detecting
peptide linkage. The intact protein yielded a
positive result since its peptide linkage is not yet
broken unlike the sample that has been
Ninhydrin test is used to detect free
alpha-amino groups. The intact protein must
yield to a positive result of yellow solution
however, due to possible experimental errors, the
result was erroneous. A positive indication of this
test for the acid hydrolysate would be a blueviolet coloration in the solution.
Xanthoproteic test is a test for presence of
aromatic rings which includes tyrosine and
tryptophan. Phenylalanine, an aromatic amino
acid, will however yield a negative result because
of inactivity. Its principle is the nitration of the
phenyl group. An orange solution yields to a
positive result which is true to the intact protein,
on the other hand, due to failure to filter the acid
hydrolysate, results were negative.
Millons test is used to detect presence of
tyrosine. Its principle is the complexation
reaction between phenolic group and mercury in
the Millons reagent. Both intact protein and the
acid hydrolysate must yield to a positive result of

red or purple-red precipitate but then again there

were errors in conducting the experiment which
affected the result.
Hopkins-Cole test detects presence of
tryptophan. Its principle is the condensation of
indole group with glyoxylic acid and sulfuric acid.
A positive result is the formation of purplecolored interface which is true to the results. Acid
hydrolysate yields to a negative result since
tryptophan cannot be detected and was
destroyed during acid hydrolysis.
Sakaguchi test detects the presence of
arginine. Its principle is the reaction of Guanido
group with a napthol and an oxidizing agent. A
positive result is a red solution which is true for
the intact protein.
Nitroprusside test is used for indicating
the presence of cysteine. A positive result is a red
solution. Its principle is complexation. Intact
protein must be very positive in this test while
the acid hydrolysate are only somewhat positive.
There must be contaminations and errors in
preparations that yielded to a negative result.
Fohls test detects sulfur-containing amino
acid and a positive result must be a black
precipitate which is true to the intact protein.
Amide test indicates primary, secondary
and tertiary amides and nitriles. A positive result
for this test is the change in color of litmus paper
from red to blue. Its principle is basic hydrolysis.
Both the intact protein and the acid hydrolysate
have positive result for this test.
Therefore casein has more than 2 peptide
bonds. It contains proline, phenylalanine,
methionine and histidine.
D. Chromatography

Figure 5. Paper chromatogram

Due to time constraint and errors in

procedure the paper chromatogram does not
chromatography results are based on the polarity
of amino acids. Tryptophan, must move farthest
from the base line since it is hydrophobic and
non-polar. On the other hand, histidine, a polar
amino acid must move least from the base line.
The mobile phase in the solvent system is the
butanol and acetic acid which means that
tryptophan has the highest affinity to the mobile
phase while histidine has a high affinity to the
stationary phase which is water.
The 1st group of amino acid, the
hydrophobic non-polar amino acids must be seen
as the farthest from the base line, while the 2 nd
and 3rd group of amino acids must be seen near
the base line which are the polar uncharged and
polar charged amino acids respectively.
Butanol, a non-polar solvent, carries the
non-polar amino acids up the chromatogram,
while the acetic acid, a polar solvent, carries the
non-polar amino acids up the chromatogram but
because of their ratio differences in the solvent
system mixture which is 4 parts of butanol for
every 1 part of acetic acid, non-polar amino acids
are favored than polar amino acids. The
Ninhydrin solution sprayed to the paper
chromatogram gave the amino acids their distinct
blue and violet colors except proline which gives
a yellow color
Amino Acid
Rf Values of the Spots
Aspartic acid
Table 2. Rf Values of the standard amino
Despite not having the solvent front to
fully reach the assigned mark, the spots on the
paper chromatogram are still semi-discernible
and can still be accounted for. The acid
hydrolysate that was used produced an orangeyellowish with shades of purple in its spotted
form, and because of the prominence of the
yellow spots, it is inferred that the acid
hydrolysate of casein contains large amount of
proline. Colors aside from yellow also indicates
presence of different amino acids.

From books
Crisostomo A. C., Daya, M. L., et al (2010)
Laboratory Manual in General Biochemistry
Quezon City: C & E Publishing, Inc.
From the Internet
Protein Purification. Retrieved March 3, 2015,
Hydrolysis of casein. Retrieved March 3, 2015,
Isoelectric Precipitation of Proteins: Casein from
Milk. Retrieved March 3, 2015, from