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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 history: The protein, Casein, was isolated from the non-fat milk using
Date Performed: December 6, 2017 isoelectric precipitation with acetic acid. Subsequent to the isolation, the
Date Submitted: December 13, 2017 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
Isoelectric precipitation qualitative color reactions. All samples gave positive results for Biuret's,
Casein Ninhydrin, Millon's and Sakaguchi tests. All of samples gave negative
Qualitative color reactions 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

1.0 Introduction can also serves as a catalysts, regulators and
protector of the body chemistry in the form
of myoglobin, hemoglobin and a variety of
Proteins, as named from the Greek
lipoproteins (Campbell et al., 2005; Nelson
word proteios meaning "first place", are
& Cox, 2013).
naturally occurring and essential in every
The process for extraction of a single
living cell. They took up almost 50% of the
type of protein from a complex mixture is
dry mass of most cells, and they are the key
referred as protein isolation. Proteins can be
instrument in almost everything a living
separated by using different methods
organism do (Campbell, Recce, Urry, &
depending on their sizes, charge, structure,
Minorsky, 2005).
hydrophobicity, and other physiochemical
It can come in different forms like in
properties. One of the most common
skin, muscles, hair, and ligaments, proteins
methods in protein isolation is isoelectric
serves as a glue that binds these together,
precipitation (Janson, 2011).
protect and give structure to the body of a
In this experiment, one protein is distilled water was added. The
isolated from milk and that is casein using concoction was mixed thoroughly. it was
the said method. The total protein content of then heated until the temperature
milk is composed of numerous specific reached the 55˚C, using a thermometer
proteins. One of the major groups of milk to monitor the temperature.
proteins are the caseins. The other major After the mixture reached the desired
milk proteins are whey proteins, beta- temperature, 10% acetic acid was added
lactoglobulin and alpha-lactalbumin, which drop wise. Consequently, the mixture
are usually synthesized in the mammary was stirred gently after every 5 drops.
epithelial cells and can only be produced by the addition of acetic acid was continued
the mammary gland (Horne, 2008). until the pH of the mixture reached 4.6
One of the significances of casein is which is monitored using a pH meter.
that it has a sufficient amino acid The congealed casein was filtered off
composition that is necessary for growth by vacuum of gravity filtration. The
and development of the nursing young. They casein residue was dried. The percent
are also highly digestible in the intestine and weight of the isolated from the powdered
are high quality source of amino acids milk was calculated.
contrary to the whey proteins that can cause
milk protein allergy since they are less 2.2 Acid and Base Hydrolysis of Intact
digestible (Horne, 2008). Protein
Casein composes the 80% of the
protein in milk. Since milk protein contains 2.2.1 Acid Hydrolysis of Intact Protein
large amounts of casein, the terms milk
protein isolation and casein isolation are In a 125-mL Erlenmeyer
used by people interchangeably. flask, a mass of 0.5-g of the isolated
The objectives of the experiment is protein was added, then a volume of
to isolate casein from the non-fat milk using 5.0-mL 8N H2SO4 was added as
isoelectric precipitation and to obtain well. A cotton plug was used as the
information about the composition of the stopper on the flask and was covered
proteins using hydrolysis and neutralization with aluminum foil. It was
which also involves qualitative color autoclaved for 5 hours at 15psi.
reactions. The hydrozylate was diluted
with 15.0-mL distilled water and the
2.0 Methodology contents were transferred into a 250-
mL beaker. The pH was checked
2.1 Isolation of Casein using pH paper and was neutralized
by adding saturated Ba(OH)2. The
A mass of 20.0-g of MilkMagic non- neutralized mixture was filtered and
fat powdered milk was placed in a 100- the filtrate was set aside to be used in
mL beaker then a volume of 50.0-mL the qualitative color reactions.
2.2.2 Alkaline Hydrolysis of Intact Protein containing the samples. It was placed in a
boiling water bath and the change in color of
In a 125-mL Erlenmeyer the mixture was noted.
flask, a mass of 0.5-g of the isolated The third test was Xanthoproteic test
protein was added, then volumes of wherein 10 drops of concentrated HNO3 is
3.0-mL boiling water and 5.0-mL added to the samples. The color of the
saturated Ba(OH)2 were added as soluttion was noted after mixing. Ten (10)
well. After labeling the flask, a drops of concentrated NaOH was then added
cotton plug was used as the stopper and the color was noted again after mixing.
on the flask and was covered with The fourth test was Millon's test
aluminum foil. It was autoclaved for wherein 5 drops of Millon's reagent was
5 hours at 15psi. added to the samples. The color of the
The hydrozylate was diluted solution was noted.
with 15.0-mL distilled water and the The fifth test was Hopkins-Cole test
contents were transferred into a 250- wherein twenty (20) drops of Hopkins-Cole
mL beaker. The pH was checked reagent was added to the samples and was
using pH paper and was neutralized mixed well. The test tube was inclined while
by adding 1.0-mL of 16N H2SO4 twenty (20) drops of concentrated H2SO4
drop wise. The neutralized mixture was added. The color of the solutions are
was filtered and the filtrate was set noted.
aside to be used in the qualitative The sixth test was Sakaguchi test
color reactions. wherein ten (10) drops of Sakaguchi reagent
was added to the samples. It was mixed and
2.3 Qualitative Color Reactions let stood for three (3) minutes. Three (3)
drops of 2% NaOBr was added and mixed.
For the sample preparation, a volume The color of the solutions aare noted.
of 1.0-mL distilled water and 0.5-g of the The last qualitative test was Fohl's
intact protein or 0.5-mL of hydrolyzed test wherein twenty (20) drops of 6M NaOH
samples were placed in a test tube. Three (3) and a few crystals of Pb(OAc)2 were added
sets of seven (7) test tubes containing intact to the samples and was mix thoroughly. It
protein, acid hydrolyzed sample, and was then heated in a boiling water bath for
alkaline hydrolyzed samples were made five (5) minutes. The color of the solutions
separately. were noted.
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
3.0 Results and Discussion 3.2 Qualitative Color Reactions

3.1 Isolation of Casein The intact protein, acid hydrolyzates

and basic hydrolyzates were all tested to
Casein is the protein that characterize and determine the functional
was isolated for the non-fat milk by adding groups that they contain. The results were
acetic acid after being heated to 55°C, to shown in Table 3, Figure 1, 2, and 3.
avoid destroying the protein (Horne, 2008). Each test that was performed has
Non-fat milk was used in the experiment to different principles behind them. The results
avoid contamination of the fats present in in the reactions of the intact proteins and
the whole milk (Minard.R., 2000). As the hydrolyzates to the reagents of each test,
acetic acid was added to the non-fat milk at vary depending on their properties. They can
a controlled pH, yellowish white coagulates, either be positive or negative in a particular
as decribed in Table 1, were produced. That test. The expected results of each tests are
process is called isoelectric precipitation. shown in Table 4.
Precipitation happened because the protein Biuret test is a general test for
has reached its isoelectric point in which its proteins and a test for detecting peptide
net charge is equal to zero (Nelson & Cox, linkage. Its principle is complexation
2013). The obtained weight per weight reaction. This complex, which is dependent
exceeded 100% because the moisture wasn't on the presence of peptide bonds, is blue-
completely removed. purple in color. The intact protein should be
For the hydrolysis of the protein, positive in this test since its peptide linkage
Ba(OH)2 was used in the alkaline hydrolysis is not broken unlike the two other samples
instead of NaOH because it is more soluble that had undergone hydrolysis (Eckersall,
in water. On the other hand, Sulfuric acid, 2008). However as seen in the results, all
H2SO4, was used in the acid hydrolysis samples gave positive result.
instead of HCl because HCl is too strong Ninhydrin test is used for detecting free
that it may cause degradation of the amino alpha amino groups and proline is the only
acids formed during hydrolysis amino acid that gives negative result for the
(Fountoulakis & Lahm, 1998; Rutherfurd & said test. Its general principle is oxidative
Gilani, 2009). Autoclaving in the hydrolysis deamination and decarboxylation. A positive
denatures the protein, it was done in order to indication of this test would be a blue violet
help the identification of the amino acid coloration in the solution (Kaiser, Colescott,
composition of the protein and act as Bossinger, & Cook, 1970). Intact protein
catalyst to accelerate the process casein and the two other hydrolyzates gave a
(Fountoulakis & Lahm, 1998). The color positive result in this test.
changes of the hydrolyzates are shown in Xanthoproteic test is used to detect
Table 2. 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
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
(, 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
clear with
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
Dark brown Yellowish brown
Sakaguchi brown Arginine is present
solution solution
Fohl’s NR NR amino acid is

Table 4 Expected Result of Samples for Each Tests

Test Intact Protein Acid Hydrolyzate Basic Hydrolyzate

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

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 Resolution Methods, and Applications:
Third Edition.
To conclude the results of the
experiment, the group has isolated the Kaiser, E., Colescott, R. L., Bossinger, C.
protein casein from the non-fat milk, D., & Cook, P. I. (1970). Color test for
however since the moisture from the isolated detection of free terminal amino groups
protein wasn't totally eliminated the in the solid-phase synthesis of peptides.
calculated percent weight of the extracted Analytical Biochemistry, 34(2), 595–
casein exceeded 100% (126% to be exact). 598.
In addition, the protein underwent acid and 2697(70)90146-6
alkaline hydrolysis after it was isolated. Minard.R. (2000). Isolation of
The intact protein and both of the Casein,Lactose and Albumin from
hydrolyzates were analyzed using qualitative Milk. Introduction to Organic
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results for Biuret's, Ninhydrin, Millon's and Approach, 16.
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negative result on Xanthoproteic test. And Lehninger Principles of Biochemistry
lastly both hydrolyzates gave negative 6th ed. Book.
results to Hopkins-cole and Fohl's tests but
the intact protein gave positives results. 6
Rutherfurd, S. M., & Gilani, G. S. (2009).
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