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The Isolation of Myoglobin from Minced Beef by Salt-Induced Precipitation and the

Determination of the Protein Concentration of Myoglobin from Minced Beef Using the
Bradford Assay
Chua, K.M.D., Cocjin, C.M.PH.R, Dizon, J.A.H., Donato, L.P.G., Dumaplin, R.A.L, Francisco,
R.A.2B-PH, Group 3, Department of Pharmacy, Faculty of Pharmacy, University of Santo
Tomas, Espaa Boulevard, 1015 Manila, Philippines
ABSTRACT
The protein, myoglobin, is found in muscle cells of animals [1].The Bradford assay is a common
method used to determine the protein concentration of a given sample [2]. This experiment was
conducted to successfully isolate the protein myoglobin from minced beef and to quantitatively
determine the protein concentration of myoglobin that was extracted from minced beef by saltinduced precipitation. The Bradford method is based on the binding of acidic Coomasie dye, a
coloring reagent, to proteins in which protein concentration is directly proportional to the dye
[3]. The standard protein that was used in this experiment is Bovine Serum Albumin (BSA)
which the Bradford assay is sensitive to than most common proteins [4]. Seven analytes were
prepared using assigned volumes of distilled water and Bovine Serum Albumin while one test
tube was left blank. The UV-Vis Spectrophotometer was used to measure the absorption of the
analytes. Apart from the nine test tubes that were prepared, another was prepared and it
contained the unknown protein. Since known concentrations of protein were prepared, the
unknown protein can be estimated and can be compared to their absorbance. To compute for the
protein concentration, the linear regression equation was used. To graphically represent the
relation of concentration and absorption, a Standard Calibration Curve was devised. It was then
used to deduce the protein concentration of the unknown sample.
INTRODUCTION
Proteins are large, complex molecule that
are composed of numerous chains of amino
acids in a specific order [5]. There are 20
amino acids that are commonly found in
proteins [2]. They are required for the
structure, function, and regulation of the
cells, tissues, and organs of the body [5].
They are classified according to their
functions. A few examples of proteins are
actin, immunoglobulin, and myoglobin [6].
Myoglobin is a protein that can be found in
the muscle cells of animals. It functions as
an oxygen-storage unit that provides oxygen
to working muscles [1].
Proteins can be isolated from a crude
sample. The most common way to

precipitate proteins is through salt-induced


precipitation [7]. At low concentrations of
salt, the solubility of the protein more often
than not increases slightly [7]. When this
happens, it is called salting in.
An assay is used to measure the
concentration of a substance; therefore, a
protein assay measures the concentration of
proteins [8]. A rapid and accurate method to
estimate protein concentration is essential in
protein studies [9]. The Coomassie blue
method otherwise known as the Bradford
method was developed by Marion M.
Bradford in 1976 [8]. The Bradford assay
relies on the binding of the dye, Coomassie

blue G-250, to protein [9]. Coomasie blue


G-250 binds to protein
given that it is in an acidic solution [8].
Considering that the Bradford assay is
colorimetric, it can be assumed that as the
protein concentration increases, the darker
the color of dye becomes. The Coomassie
dye has a maximum absorbance of 590nm
[9]. Hence, the quantity of protein can be
estimated by determining the amount of dye
in the blue ionic form [9]. This is frequently

achieved by measuring the absorbance of the


solution at 595 nm [9].

METHODOLOGY

transferred into a new beaker using filter


paper. The extract was divided and put in
separate centrifuge tubes. The individual test
tubes containing the extract were
centrifuged at 13,000 x g for five minutes.
After being centrifuged, 1.5 mL of the
supernatant was transferred to another
beaker. Approximately, 0.30-0.35 g of
ammonium sulfate [(NH4)2SO4] crystals
were ground into a fine powder and was
added to the supernatant. It was mixed
gently until the powder was entirely
dissolved in the supernatant. Frothing was
avoided. The sample was again centrifuged
at 13,000 x g for five minutes. The
supernatant was then decanted, leaving only
myoglobin. The myoglobin was set aside.

For the isolation of myoglobin, the


apparatus that were used were a centrifuge, a
graduated cylinder, a triple beam balance, a
watch glass, a stirring rod, Pasteur pipettes,
beakers, a funnel, filter paper, and centrifuge
tubes. The reagents and materials that were
used were minced beef, ammonium sulfate
[(NH4)2SO4] crystals, 70% buffer-diluted
ammonium sulfate [(NH4)2SO4] solution
with a pH of 7.5. For the protein assay, the
apparatus that were used were, test tubes, a
test tube rack, pipette, rubber aspirator, tape,
a
stirring
rod,
and
a
UV-Vis
Spectrophotometer. The reagents that were
used for the assay were, the Bradford
reagent, Bovine serum albumin standard
(BSA), and the evaporated milk sample.
Six grams of minced beef was weighed on a
triple beam balance with the aid of a preweighed watch glass. Six milliliters of the
70% ammonium sulfate [(NH4)2SO4]
solution was measured using a graduated
cylinder. The minced beef and the 70%
ammonium sulfate solution were placed
together in a beaker. The mixture was stirred
for one minute to allow the release of
myoglobin. The dark-red extract was

The objectives of this experiment are: (1) to


isolate myoglobin from minced beef by saltinduced
precipitation
and
(2)
to
quantitatively determine the protein
concentration of a given sample through
Bradford assay.

After the reagents and materials for the


Braford assay were prepared, a series of test
tubes were prepared with containing the
following:
Tube
No.

10

mL
distilled
H2O
mL
Standar
d BSA

2.
5

2.0

2.0

1.5

1.5

1.0

1.0

0.5

0.5

2.
0

0.5

0.5

1.0

1.0

1.5

1.5

2.0

2.0

0.
5

There were four standards that were


prepared by the class. Two groups were
assigned per standard. The first test tube was
left blank; while the last test tube contained
the unknown sample. Subsequently, 2.5 mL
of Bradford reagent was added to all 10 test
tubes. The mixtures were mixed using a
stirring rod and they were placed in a test
tube rack to stand for 5 minutes. The
absorbance was read at 595 nm within the
hour. The albumin standard curve was
constructed by plotting the A595 against the
concentration
(mg/mL).
The
linear
regression was also used to compute for the
concentration.
RESULTS AND DISCUSSION
Myoglobin is an oxygen-carrying and
storage protein of muscle that resembles
hemoglobin but contains only one subunit
and one heme as part of the molecule (rather
than the four of hemoglobin), and with a
molecular weight approximately one quarter
than that of hemoglobin [10].
The protein, myoglobin, can be extracted
from the minced beef using the method
called salt-induced precipitation. Saltinduced precipitation is the most common
way to precipitate proteins [7]. Due to the
bad quality of the beef that was used in the
experiment, the group failed to extract
myoglobin from the beef. This was proven
using the different qualitative color
reactions. Despite this, the group still
performed all the tests on the sample.
The Bradford assay is a fast, convenient, and
efficient way of determining the amount of
protein present in a solution [4]. The
Coomasie dye that is present in the Bradford
reagent shifts from 465 nm to 595 nm when

protein is bound to the dye [4]. The more


protein is present in a solution, the darker
the dye got. A UV-Vis Spectrophotometer is
used to measure the how much light was
absorbed by the samples. Two trials were
done and the average of the two values were
the ones that were used in the graph.
However, time was scarce and the class was
not
able
to
use
the
UV-Vis
Spectrophotometer. Instead, the instructor
provided the class the data. The data is
tabulated as seen below.
TABLE 1. DATA COLLECTED PROVIDED
Sample

Total protein
concentration
(mg/mL)

A595
Trial 1

A595
Trial 2

A595
Average

Blank

Standard
1
Standard
2
Standard
3
Standard
4
Unknow
n

0.050

0.2485

0.2265

0.2375

0.040

0.2245

0.2036

0.2141

0.030

0.1864

0.1910

0.1887

0.020

0.1529

0.1512

0.1521

0.1421

The data above will be used to make the


standard curve graph with protein
concentration as the x-axis and the A595 as
the y-axis.
Standard Curve Graph
0.4
A595 0.2
0

0.01 0.02 0.03 0.04 0.05 0.06


Protein Concentration

To get the A595 of the unknown protein, the


linear regression equation was used. The
formula is seen in the figure below.
FIGURE 1: LINEAR REGRESSION EQUATION

Linear regression ( y )=a+ bx

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Medical Dictionary. (2012). Retrieved
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