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Airika P. Muhi, Charlot P. Navarro, Kristin D. Oanes,

Jhunabelle D. Pablo, Irish Jane M. Patron, Margaret Corinne U. Ramos
Group 6
2G Pharmacy
PH Biochemistry Laboratory

Different proteins exist in a single cell. Many techniques are performed to eliminate contaminants and to arrive at a
pure sample of the protein of interest. The main objective of this experiment is to extract particular proteins from
certain substances namely: (a) Casein from skimmed milk by isoelectric precipitation; (b) Albumin from skimmed milk
by heat denaturation; (c) Gluten from wheat flour by difference in solubility; and (d) Myoglobin from beef steak by
salt-induced precipitation. In this experiment, the group is assigned to use beef steak in order to extract the protein
myoglobin. Protein precipitation is utilized in processing biological products in concentrating proteins and to purify
them from contaminants. Salting out is the process in which the desired concentration of the salt is achieved in the
solution. Myoglobin was isolated by ammonium sulfate precipitation form the buffered muscle extract. The experiment
resulted to a reddish-brown liquid which was extracted from the meat. The color shows that myoglobin contains
hemes which are responsible for its red color. It also indicates the degree of oxidation in the protein myoglobin. After
isolation, the intact protein was tested for color reactions using different reagents and solutions. The protein was
tested for the following reactions: Biuret, Ninhydrin, Xanthoproteic, Millons, Hopkins-Cole, Sakaguchi, Nitroprusside,
Fohls, Test for Amides and Pauly. The tests resulted to different colors which indicated the amino acids present in the

In the isolation of an intact protein, the following
are considered: (a) three-dimensional structure
of the protein (fibrous or globular); (b)
interactions that keep te native conformation of
the protein functional (electrostatic, covalent,
hydrophobic, H-bonding, and Van der Waals); (c)
Acid-base property; and (d) Solubility of protein
in different solvents. The solubility of proteins are
altered through changing the pH of their
environment. Denaturation of the proteins is
essential to disrupt the native conformation of
the proteins. It alters the proteins function,
demonstrating relationship between structure and
function. Particular proteins are extracted from
different substances and by different processes.
In this experiment, Gluten was extracted from
flour, Casein and Albumin were extracted from
milk and Myoglobin was extracted from beef
It was assigned to the group to extract the
protein myoglobin from beef steak. Myoglobin is
the classic example of a globular protein. It is an
oxygen-carrying protein found in muscle tissue.
It is the protein that is responsible for transport
and storage of oxygen in higher organisms[1].
The complete myoglobin molecule consists of a
single polypeptide chain of 153 amino acid
residues and includes a prosthetic group, the
heme group, which also occurs in hemoglobin[2].
The heme group is also an essential component
of this protein. The Fe2+ ion in the heme group is
the binding site for oxygen in both myoglobin and

hemoglobin. However, myoglobin has greater

attraction for oxygen than does hemoglobin,
which allows efficient transfer of oxygen from the
bloodstream to the cells of the body[1].
Myoglobin was isolated in this experiment by
ammonium sulfate precipitation. Ammonium
sulfate is the most common reagent to use at this
step and this procedure is referred to as salting
Amino acids have a variety of chemically
reactive groups. The reactions for side chains, amino acid, and -carboxyl groups can be used
to characterize both free amino acids and
proteins[3]. Another objective in this experiment
is to analyze the chemical groups on the protein
myoglobin which responsible for color reactions
and to explain the principles involved in each

A. Compounds tested
Beef steak, Isolated protein
B. Procedure
Isolation of Myoglobin
1. 6.0 g of minced heart steak was placed in
a small beaker. Then, 6 ml of 70%
(NH4)2SO4 was added.

2. The mixture was gently stirred for 1

minute to release the myoglobin.
3. The dark-red extract was expressed in a
beaker using a cheesecloth.
4. The extract was centrifuged at 13,000 x g
for 5 minutes.
5. 1.5 ml of the supernatant was transferred
into another empty centrifuge tube.
6. 0.30 0.35 g of (NH4)2SO4 crystals which
was ground to fine powder was added.
The solution was gently mixed until the
solid was dissolved. Frothing was avoided.
7. The sample was centrifuged for at least 5

8. The supernatant was decanted.

Hopkins-Cole test
1. 20 drops of Hopkins-Cole reagent was
added to the samples.
2. 20 drops of conc. H2SO4 was slowly added
to the test tube in an inclined manner. The
solution was avoided to be mixed.
Sakaguchi test
1. 10 drops of 10% NaOH and 10 drops of
0.02% naphthol solution was added to the
sample. Soluton was mixed and stand for
3 minutes.
2. 3 drops of NaOBr was added.
Nitroprusside test

Color Reactions of Intact Protein

10 test tubes were prepared with the intact
protein solution and a small amount of distilled
water is added to each sample.
Biuret test
1. 20 drops of 2.5 M NaOH was added to the
2. 2-3 drops of 0.1 M CuSO4 was added to
the solution.
3. The test tube was shaken and the color
was noted.
Ninhydrin test
1. 6-10 drops of 0.1% ninhydrin solution into
the diluted samples.
2. The tube was heates in a boiling water
bath. The appearance of a blue-violet
coloration was noted.
Xanthoproteic test
1. 10 drops of conc. HNO3 was slowly added
to the diluted samples. The solution was
2. 10 drops of conc. NaOH was slowly added.
The solution was mixed.
Millons test
1. 5 drops of Millons reagent was added to
the diluted samples.
2. The change in color was noted.

1. 0.5 ml of 3 M NaOH was added to 0.5 ml

of sample.
2. 0.25 ml of nitroprusside solution was
3. The formation of a red solution was noted.
Fohls test
1. 5 drops of 30% NaOH and 2 drops of 5%
(CH3COO)2Pb was added to the samples.
2. The tube was placed in a boiling water
3. The appearance of dark (black or brown)
sediment was noted.
Test for Amides
1. 1ml of 20% NaOH to 10 drops of the
2. The tube was placed in a boiling waster
3. The evolution of gas was tested during
heating by placing a moistened red litmus
paper over the mouth of the tube.
Pauly test
1. The diazo reagent was prepared by mixing
3-5 drops of 1% sulfanillic acid with 3
drops of 5% NaNO2.
2. 5 drops of the sample and 3-5 drops of
10% Na2CO3 was added to the diazo


Isolation of Myoglobin
The protein must first be released from the
cells and subcellular organelles. Separation
techniques focus on size, chrge and polarity.
Homogenization is the first step which involves
breaking open the cells[2]. In the case of
myoglobin, denaturation which is the unfolding of
the protein is used to disrupt its native
conformation. The beef steak was minced to
reduce the size which will help for the easier
extraction of the protein.

Table 1. Isolation of Proteins


White, rubb
White semis
White amor
Reddish bro

The extracted produced a liquid with a reddish

brown color. The color indicated the presence of
hemes in the protein myoglobin. It also shows
the degree of oxidation in the protein.
Color reactions of Intact Protein
There are many variety of amino acids which
contain chemically reactive groups. The protein is
tested for the presence of different amino acids
which is indicated by different colors.

Figure 1. Structure of Hemoglobin

After adding the (NH4)2SO4 solution the
resulting extract is still not pure. The muscle was
buffered then the next method was done which is
the salting out. This method is commonly used to
precipitated the desired protein. (NH4)2SO4
crystals was added to the extract.
After the proteins are solubilized, they are
often subjected to crude purification based on
solubility. Ammonium sulfate is the most common
reagent to use at this step. When ammonium
sulfate is added to the protein solution, some of
the water is taken away from the protein to make
ion-dipole bond with the salts. With less water
available to hydrate the proteins, they begin to
interact with each other through hydrophobic
bonds. At a defined amount of ammonium
sulfate, a precipitate contains contaminating
protein forms. These proteins are centrifuged
down and discarded. Then more salt is added,
and a different set of proteins which usually
contains the protein desired precipitates. This
precipitate is collected by centrifugation and

The biuret test is used to detect the presence

of peptide bonds. The reagents used in this test
are NaOH and CuSO4. It yielded with a blue color.
The Ninhydrin test is a test for an -amino
acid. A deep blue or purple color known as
Ruhemanns purple is produced in this test.
However, in the experiment it resulted a colorless
Xanthoproteic test indicates side chains of
aromatic amino acids. The test is positive if the
color produced is yellow or dark yellow. But, in
the experiment it yielded again a colorless
Millons test determines tyrosine residues. A
reddish-brown coloration indicates presence of
tyrosine. In the experiment it gave a negative
reaction because the solution produced was
Hopkins-Cole test indicates the presence of
tryptophan residues. It is indicated by a purple
ring. There is no purple ring appearance on the
solution so it does not contain tryptophan.
Sakaguchi test detects the presence of
arginine which contains the guanido group. The
reagents used were NaOH, naphthol solution and
NaOBr. It resulted to a colorless solution.


The Nitroprusside test is utilized in looking for

sulfur-containing amino acids present.

Proteins with free thiol group usually give a color

of red. In this experiment, the solution produces
was salmon pink in color.
Fohls test is used to identify the presence of
Methionine and Glutamic acid. It is indicated by a
black or brown sediment. There is no presence of
brown sediment on the experiment done.
The test for amides is used to indicate the
presence of R-groups of asparagines and
glutamine. The litmus paper turned blue from
Pauly test indicates the presence of tyrosine
or histidine. It is expected to yield a red solution
but the solution produced in the experiment is
Table 2. Color reactions of Intact protein
Color reaction

Intact protein
No presence of violet
Salmon pink
No presence of brown

Test for Amide


Red to blue on litmus

Out of all the color reactions, the closest

positive reactions were yielded from the Biuret
and Nitroprusside which indicates the presence of
proteins or polypeptides and sulfur-containing
amino acid, respectively.

[1] Campbell, M. K. & Farrell, S. O. (2012).
Biochemistry, 7th ed. International Edition.
China: China Translation & Printing
Services Limited
[2] Caret, R. L., et. al. (2008). General,
Organic, and Biochemistry 6th ed.
New York: The McGraw-Hill Companies,
[3] Crisostomo, A.C., et. al. (2010).
Laboratory Manual in General Biochemistry.
Quezon City: C & E Publishing, Inc.