Formal Report
Experiment No. 2
Isolation and Characterization of Proteins
Domingo, A.J.A.1
1Chemistry Department, College of Science, Adamson University, Ermita, Manila 1000 Philippines
ARTICLE INFO
Article history:
Date Performed: December 6, 2017
Date Submitted: December 13, 2017
Keywords:
Isoelectric precipitation
Casein
Qualitative color reactions
1.0 Introduction
Proteins, as named from the Greek
word proteios meaning "first place", are
naturally occurring and essential in every
living cell. They took up almost 50% of the
dry mass of most cells, and they are the key
instrument in almost everything a living
organism do (Campbell, Recce, Urry, &
Minorsky, 2005).
It can come in different forms like in
skin, muscles, hair, and ligaments, proteins
serves as a glue that binds these together,
protect and give structure to the body of a
ABSTRACT
The protein, Casein, was isolated from the non-fat milk using
isoelectric precipitation with acetic acid. Subsequent to the isolation, the
protein underwent acid and alkaline hydrolysis. The intact protein and
both acid and basic hydrolyzates were subjected to various qualitative
color reactions namely, Biuret test, Ninhydrin test, Xanthoproteic test,
Millon’s test, Hopkins-Cole test, Sakaguchi test, and Fohl’s test. The
intact protein and both of the hydrolyzates were analyzed using
qualitative color reactions. All samples gave positive results for Biuret's,
Ninhydrin, Millon's and Sakaguchi tests. All of samples gave negative
result on Xanthoproteic test. And lastly both hydrolyzates gave negative
results to Hopkins-cole and Fohl's tests but the intact protein gave
positive results.
multi-celled organism. On the other hand, it
can also serves as a catalysts, regulators and
protector of the body chemistry in the form
of myoglobin, hemoglobin and a variety of
lipoproteins (Campbell et al., 2005; Nelson
& Cox, 2013).
The process for extraction of a single
type of protein from a complex mixture is
referred as protein isolation. Proteins can be
separated by using different methods
depending on their sizes, charge, structure,
hydrophobicity, and other physiochemical
properties. One of the most common
methods in protein isolation is isoelectric
precipitation (Janson, 2011).
 In this experiment, one protein is
isolated from milk and that is casein using
the said method. The total protein content of
milk is composed of numerous specific
proteins. One of the major groups of milk
proteins are the caseins. The other major
milk proteins are whey proteins, beta-
lactoglobulin and alpha-lactalbumin, which
are usually synthesized in the mammary
epithelial cells and can only be produced by
the mammary gland (Horne, 2008).
One of the significances of casein is
that it has a sufficient amino acid
composition that is necessary for growth
and development of the nursing young. They
are also highly digestible in the intestine and
are high quality source of amino acids
contrary to the whey proteins that can cause
milk protein allergy since they are less
digestible (Horne, 2008).
Casein composes the 80% of the
protein in milk. Since milk protein contains
large amounts of casein, the terms milk
protein isolation and casein isolation are
used by people interchangeably.
The objectives of the experiment is
to isolate casein from the non-fat milk using
isoelectric precipitation and to obtain
information about the composition of the
proteins using hydrolysis and neutralization
which also involves qualitative color
reactions.
2.0 Methodology
2.1 Isolation of Casein
A mass of 20.0-g of MilkMagic non-
fat powdered milk was placed in a 100-
mL beaker then a volume of 50.0-mL
distilled water was added. The
concoction was mixed thoroughly. it was
then heated until the temperature
reached the 55˚C, using a thermometer
to monitor the temperature.
After the mixture reached the desired
temperature, 10% acetic acid was added
drop wise. Consequently, the mixture
was stirred gently after every 5 drops.
the addition of acetic acid was continued
until the pH of the mixture reached 4.6
which is monitored using a pH meter.
The congealed casein was filtered off
by vacuum of gravity filtration. The
casein residue was dried. The percent
weight of the isolated from the powdered
milk was calculated.
2.2 Acid and Base Hydrolysis of Intact
Protein
2.2.1 Acid Hydrolysis of Intact Protein
In a 125-mL Erlenmeyer
flask, a mass of 0.5-g of the isolated
protein was added, then a volume of
5.0-mL 8N H2SO4 was added as
well. A cotton plug was used as the
stopper on the flask and was covered
with aluminum foil. It was
autoclaved for 5 hours at 15psi.
The hydrozylate was diluted
with 15.0-mL distilled water and the
contents were transferred into a 250-
mL beaker. The pH was checked
using pH paper and was neutralized
by adding saturated Ba(OH)2. The
neutralized mixture was filtered and
the filtrate was set aside to be used in
the qualitative color reactions.
2.2.2 Alkaline Hydrolysis of Intact Protein
In a 125-mL Erlenmeyer
flask, a mass of 0.5-g of the isolated
protein was added, then volumes of
3.0-mL boiling water and 5.0-mL
saturated Ba(OH)2 were added as
well. After labeling the flask, a
cotton plug was used as the stopper
on the flask and was covered with
aluminum foil. It was autoclaved for
5 hours at 15psi.
The hydrozylate was diluted
with 15.0-mL distilled water and the
contents were transferred into a 250-
mL beaker. The pH was checked
using pH paper and was neutralized
by adding 1.0-mL of 16N H2SO4
drop wise. The neutralized mixture
was filtered and the filtrate was set
aside to be used in the qualitative
color reactions.
2.3 Qualitative Color Reactions
For the sample preparation, a volume
of 1.0-mL distilled water and 0.5-g of the
intact protein or 0.5-mL of hydrolyzed
samples were placed in a test tube. Three (3)
sets of seven (7) test tubes containing intact
protein, acid hydrolyzed sample, and
alkaline hydrolyzed samples were made
separately.
For the Biuret test, ten (10) drops of
Biuret reagent was added to the test tube
containing the samples. It was shook and the
color change was noted.
The second test was Ninhydrin test
wherein 6-10 drops of 0.1% Ninhydrin
solution was added to the test tube
containing the samples. It was placed in a
boiling water bath and the change in color of
the mixture was noted.
The third test was Xanthoproteic test
wherein 10 drops of concentrated HNO3 is
added to the samples. The color of the
soluttion was noted after mixing. Ten (10)
drops of concentrated NaOH was then added
and the color was noted again after mixing.
The fourth test was Millon's test
wherein 5 drops of Millon's reagent was
added to the samples. The color of the
solution was noted.
The fifth test was Hopkins-Cole test
wherein twenty (20) drops of Hopkins-Cole
reagent was added to the samples and was
mixed well. The test tube was inclined while
twenty (20) drops of concentrated H2SO4
was added. The color of the solutions are
noted.
The sixth test was Sakaguchi test
wherein ten (10) drops of Sakaguchi reagent
was added to the samples. It was mixed and
let stood for three (3) minutes. Three (3)
drops of 2% NaOBr was added and mixed.
The color of the solutions aare noted.
The last qualitative test was Fohl's
test wherein twenty (20) drops of 6M NaOH
and a few crystals of Pb(OAc)2 were added
to the samples and was mix thoroughly. It
was then heated in a boiling water bath for
five (5) minutes. The color of the solutions
were noted.
3.0 Results and Discussion
3.1 Isolation of Casein
Casein is the protein that
was isolated for the non-fat milk by adding
acetic acid after being heated to 55°C, to
avoid destroying the protein (Horne, 2008).
Non-fat milk was used in the experiment to
avoid contamination of the fats present in
the whole milk (Minard.R., 2000). As the
acetic acid was added to the non-fat milk at
a controlled pH, yellowish white coagulates,
as decribed in Table 1, were produced. That
process is called isoelectric precipitation.
Precipitation happened because the protein
has reached its isoelectric point in which its
net charge is equal to zero (Nelson & Cox,
2013). The obtained weight per weight
exceeded 100% because the moisture wasn't
completely removed.
For the hydrolysis of the protein,
Ba(OH)2 was used in the alkaline hydrolysis
instead of NaOH because it is more soluble
in water. On the other hand, Sulfuric acid,
H2SO4, was used in the acid hydrolysis
instead of HCl because HCl is too strong
that it may cause degradation of the amino
acids formed during hydrolysis
(Fountoulakis & Lahm, 1998; Rutherfurd &
Gilani, 2009). Autoclaving in the hydrolysis
denatures the protein, it was done in order to
help the identification of the amino acid
composition of the protein and act as
catalyst to accelerate the process
(Fountoulakis & Lahm, 1998). The color
changes of the hydrolyzates are shown in
Table 2.
3.2 Qualitative Color Reactions
The intact protein, acid hydrolyzates
and basic hydrolyzates were all tested to
characterize and determine the functional
groups that they contain. The results were
shown in Table 3, Figure 1, 2, and 3.
Each test that was performed has
different principles behind them. The results
in the reactions of the intact proteins and
hydrolyzates to the reagents of each test,
vary depending on their properties. They can
either be positive or negative in a particular
test. The expected results of each tests are
shown in Table 4.
Biuret test is a general test for
proteins and a test for detecting peptide
linkage. Its principle is complexation
reaction. This complex, which is dependent
on the presence of peptide bonds, is blue-
purple in color. The intact protein should be
positive in this test since its peptide linkage
is not broken unlike the two other samples
that had undergone hydrolysis (Eckersall,
2008). However as seen in the results, all
samples gave positive result.
Ninhydrin test is used for detecting free
alpha amino groups and proline is the only
amino acid that gives negative result for the
said test. Its general principle is oxidative
deamination and decarboxylation. A positive
indication of this test would be a blue violet
coloration in the solution (Kaiser, Colescott,
Bossinger, & Cook, 1970). Intact protein
casein and the two other hydrolyzates gave a
positive result in this test.
Xanthoproteic test is used to detect
for the presence of aromatic rings which
includes tryptophan and tyrosine. Although
phenylalanine is considered one of them, it
will not have a positive result because it is oxidizing reagent. A positive indication for
inactive. Its principle is the nitration of the this test is a red or orange solution. As seen
phenyl group. A positive indication for this in the results, all sample gave positive
test is a yellow to orange coloration solution. results (Tomlinson & Viswanatha, 1974).
Intact proteins, acid hydrolyzates and basic All the arginine in the basic hydrolyzate
hydrolyzates should be positive for this test wasn't destroyed during the hydrolysis.
(Sim, Chin, Tso, & Thong, 2008). However, The last test, Fohl’s test is used for
in the result for this test, all samples gave the detection of sulfur containing proteins. It
negative results. Sources of errors could also indicates the presence of methionine
possibly be the preparation and execution of and cysteine because those two amino acids
the test. have sulfur in their structures. Its principle is
Millon’s test is used for the detection fusion followed by ionic interaction. A
of the presence of tyrosine. Its principle is positive result for this test is the formation
the complexation reaction between phenolic of black precipitate from lead sulfide (PbS)
group and mercury that is found in the (Wang, Su, Jia, & Jin, 2013). All samples
Millon’s reagent. A positive indication of should gave a positive results however only
this test is red precipitate (Sim et al., 2008). the intact protein showed dark coloration.
Intact proteins and the hydrolyzates gave
positive results in this test. Table 1. Isolation of Casein
Hopkins-Cole test is used for the
Proteins Description
detection of the presence of tryptophan. Its yellowish white
principle is the condensation of indole group Casein %w/w: 126
precipitate
with glyoxylic acid and H2SO4. A positive
indication of this test is the formation of Table 2 Hydrolysis of Intact Protein
purple ring on the surface of the solution
(vlab.amrita.edu, 2011). Only the acid Description of
hydrolyzate should be negative for this test Mode of Hydrolysate
Hydrolysis (autoclaving)
because tryptophan cannot be detected and
was destroyed during acid hydrolysis. Before After
However all of the samples gave negative White Yellow
results, errors were made to cause this solids with solution
Acid
clear with
outcome.
solution precipitate
Sakaguchi test is used for the White
detection of the presence of free or intact solids with Yellow
arginine. Since alkaline or basic hydrolysis Alkaline
clear solution
destroys arginine and produces ornithine and solution
urea, basic hydrolyzate must be negative for
this test while all the other samples are
positive. Its principle is the reaction of
Guanido group with α napthol and an
Table 3 Results of Qualitative Color Reactions

Intact Protein Acid Basic

Test Inference
(casein) Hydrolyzate Hydrolyzate
Purple Peptide linkage is
Biuret Purple solution Pink solution
solution present
Blue Blue violet Free α amino group
Ninhydrin Violet solution
coloration solution is present
No aromatic amino
Xanthoproteic NR NR NR
acid is present
Red orange Red orange Orange
Millon’s Tyrosine is present
precipitate precipitate precipitate
Violet Tryptophan is
Hopkins-Cole No purple ring No purple ring
interference present
Yellowish
Dark brown Yellowish brown
Sakaguchi brown Arginine is present
solution solution
solution
Sulfur-containing
Black
Fohl’s NR NR amino acid is
precipitate
present

Table 4 Expected Result of Samples for Each Tests

Test Intact Protein Acid Hydrolyzate Basic Hydrolyzate

(casein)
Biuret + - -
Ninhydrin + + +
Xanthoproteic + + +
Millon’s + + +
Hopkins-Cole + - +
Sakaguchi + + -
Fohl’s + + +

Table 5 Actual Results of the Samples for Each Tests

Test Intact Protein Acid Hydrolyzate Basic Hydrolyzate

(casein)
Biuret + + +
Ninhydrin + + +
Xanthoproteic - - -
Millon’s + + +
Hopkins-Cole + - -
Sakaguchi + + +
Fohl’s + - -
 Figure 1 Intact Protein Qualitative Color Reaction Results

Figure 2 Acid Hydrolyzate Qualitative Color Reaction Results

Figure 3 Basic Hydrolyzate Qualitative Color Reaction Results

4.0 Conclusion
To conclude the results of the
experiment, the group has isolated the
protein casein from the non-fat milk,
however since the moisture from the isolated
protein wasn't totally eliminated the
calculated percent weight of the extracted
casein exceeded 100% (126% to be exact).
In addition, the protein underwent acid and
alkaline hydrolysis after it was isolated.
The intact protein and both of the
hydrolyzates were analyzed using qualitative
color reactions. All samples gave positive
results for Biuret's, Ninhydrin, Millon's and
Sakaguchi tests. All of samples gave
negative result on Xanthoproteic test. And
lastly both hydrolyzates gave negative
results to Hopkins-cole and Fohl's tests but
the intact protein gave positives results.
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