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ABSTRACT
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
protein.
INTRODUCTION
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
muscle[3].
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
EXPERIMENTAL
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.
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
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.
Descriptio
White, rubb
White semis
White amor
Reddish bro
the
Intact protein
Blue
Colorless
Colorless
Colorless
No presence of violet
ring
Colorless
Salmon pink
No presence of brown
sediment
Red to blue on litmus
paper
Orange
REFERENCES
[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,
Inc.
[3] Crisostomo, A.C., et. al. (2010).
Laboratory Manual in General Biochemistry.
Quezon City: C & E Publishing, Inc.