Biochemistry Laboratory (BCM 362L), Experiment No.

3, © February 14, 2006

3nd Quarter A.Y. 2005-2006

Extraction and Characterization of Proteins

Mr. *****1, *****2, *****2

1
Professor, School of CHE-Chm, Mapua Institute of Technology
2
Student, BCM 362L/ A31, School of CHE-Chm, Mapua Institute of Technology
______________________________________________________________________________________

ABSTRACT

Invertase, Albumin and Casein were extracted By using the Warburg-Christian method, the
from yeast, egg and milk respectively using concentration of casein is 0.3329 mg/mL.
solvent extraction, salting out and isoelectric Different concentrations for different absorbance
precipitation. Benedict’ test was performed to was computed using Beer’s law for the Bradford
determine the presence of reducing sugars in the assay method, a linear plot was calibrated.
prepared solution. Concentrations of albumin
and casein were determined by two methods – Keywords: extraction, assay, isoelectric,
Warburg-Christian method and Bradford Assay. calibrated.
______________________________________________________________________________________

INTRODUCTION that it is direct and it is nondestructive. The

Proteins are the most diverse and
abundant macromolecules within cells. Their
functions range from the enzymes that carry out
the numerous metabolic processes of the cell to
structural components that gives cells structure
and organization. One of the most useful
parameters used in the study of proteins in
solution is the concentration of the protein and. It
is necessary for biochemists to estimate the
concentration of proteins in a solution of interest
in order to quantitatively determine the activity
of the protein.
In one part of the experiment,
Benedict’s test was used. Here, monosaccharides
and disaccharides can be detected because of
their free aldehyde groups, thus, testing positive
for the Benedict's test. Such sugars act as a
reducing agent, and is called a reducing sugar.
By mixing the sugar solution with the Benedict's
solution and adding heat, an oxidation-reduction
reaction will occur. The sugar will oxidize,
gaining an oxygen atom, and the Benedict's
reagent will reduce, loosing an oxygen atom.
There are two types of
spectrophotometric methods for determining
protein concentration, which will be used for
albumin and casein. The first method is called
the Warburg-Christian method. In this method
the protein concentration is estimated by making
a direct absorption measurement of a solution in
the UV range. The advantage of this method is
method also has disadvantages. First, the
absorption at 280 nm will vary from protein to
protein depending on the content of tyrosine and
tryptophan.. Therefore, absolute quantitation of
protein concentration by this method can only be
determined for pure preparations of protein.
Second, any substance that absorbs at 280 nm
will interfere with the reading. However, the
method can be quite useful as a rapid and
nondestructive method to compare the relative
concentrations of numerous protein solutions,
such as the fractions generated during protein
purification by column chromatography. The
sensitivity of the assay varies with the tyrosine
and tryptophan content of the protein solution,
but is in the range of 1 mg protein/ml to 1 mg
protein/ml.
The second method to determine protein
concentration is the protein dye-binding assay or
Bradford assay. The dye, Coomasie brilliant
blue, is a relatively hydrophobic substance that
binds avidly to the hydrophobic regions of
proteins. The change in absorption can be used
to quantitate indirectly the amount of protein in
the solution. The color change can be quantified
with a spectrophotometer. There are two major
advantages to the Bradford assay: 1) the ease of
performance and 2) few interfering substances.
The only interfering substances are high
concentrations of detergents that disrupt the
binding of the dye to protein because of their
amphipathic nature. The sensitivity of the
Bradford assay is in the range of 1 mg to 100 mg
of protein.3, 4
METHODOLOGY
The proteins invertase, albumin and
casein were prepared from yeast, egg and milk
respectively for analysis.
Invertase from Yeast
A clean mortar and pestle was prepared
and to it 10 g of baker’s yeast with 5 g of sand
was grinded until the fine powder was obtained.
10 mL of hexane was added to this fine powder.
30 mL of water in 3 mL portions was added and
the grinding was continued for 15 minutes. A
cell free extract was prepared by filtering the
grounded yeast through cheesecloth. The filtrate
was centrifuged at 6000 rpm for 5 minutes and
the sediment was discarded. The centrifugation
step was repeated to get the clear supernatant.
The supernatant was poured into a 50-mL beaker
and 200 mL of 95% ethanol was slowly added. It
was set in an ice bath until the precipitation had
occurred. The resulting suspension was
centrifuged at 3000 rpm for 3 minutes. The
supernatant was discarded. The invertase crude
extract will be used for the analysis.
Four test tubes were set according to the
table below:
Volume of solutions (mL)
Test
0.3 M 2% 2%
tube H20 Buffer
#
Sucrose Glucose Fructose
1 6.0 4.0 6.0 - -
2 6.0 8.0 6.0 - -
3 - 8.0 6.0 1.0 1.0
4 - 10.0 6.0 - -
The test tubes were placed in a water
bath which was maintained at 37ºC and
equilibrated for 10 minutes. 4 mL of invertase
extract was added to test tubes 1, 3, 4. The tubes
were kept in the water bath for 6 minutes more.
The reaction was stopped by adding 2 mL of
10% NaOH to the test tubes. The presence of
reducing sugars was tested using Benedict’s
reagent.
Albumin from Egg
30 mL of egg white was measured and
was placed in a beaker. The egg white was
stirred with a stirring rod and 3 mL of 1.0 M
HOAc was added. The resulting mixture was
filtered through the cheesecloth by stirring
vigorously with a glass rod to speed up the
passage of the filtrate through the cheesecloth
and was squeezed to break the membranes. The
filtrate was collected in a 250-mL beaker. An
equal volume of saturated (NH4)2SO4 solution
was added to the filtrate. The mixture was
allowed to stand for 30 minutes. The mixture
was centrifuged and the precipitate was
discarded. The supernatant was transferred into
a 250-mL Erlenmeyer flask. (NH4)2SO4 buffer
was added to the clear yellow supernatant while
it was continuously stirring until turbidity
persists. The mixture was then cooled for two
days to allow precipitation of albumin. The
mixture was centrifuged and the resulting
supernatant was discarded while the precipitate
was collected. The crude precipitate was
weighed. A 10% solution of albumin in 0.9%
NaCl was prepared.
Casein from Milk
25 mL of whole milk was prepared by
diluting it with 75 mL of skim milk. The
prepared milk was heated to 40ºC while
constantly stirring. 0.1 M HCl was added drop-
wise over a period of 10 minutes until a
flocculent precipitate forms. The curd was
allowed to settle and the whey was decanted.
The curd was then washed by resuspending it in
15 mL of water while stirring vigorously.
RESULTS AND DISCUSSION
Benedict’s test
Yeast (Saccharomyces cerevisiae)
invertase is essentially an intracellular enzyme
and disruption of cell membranes is necessary
for its extraction. The crude extract, invertase,
prepared by autolysis of dried baker's yeast gives
satisfactory activity with no interfering activities.
The yeast enzyme invertase is a b-
fructofuranosidase that can hydrolyze sucrose
which is D-glucopyranosyl (12)-b-D-
fructofuranoside into glucose and fructose.
Generally, invertase acts as a catalyst to convert
sucrose into glucose and fructose.
A simple test using Benedict’s reagent
containing a solution of copper sulfate, sodium
hydroxide, and tartaric acid was performed to
indicate the presence of reduced sugar in the test Bradford Assay
tubes. The Bradford assay is a very popular
protein assay method because it is simple, rapid,
Test tube # Description inexpensive and sensitive. The Bradford assay
works by the action of Coomassie brilliant blue
1 (+) – reddish brown
G-250 dye (CBBG). This dye specifically binds
2 (+) – light reddish brown
to proteins at arginine, tryptophan, tyrosine,
3 (+) – dark reddish brown
histidine and phenylalanine residues. CBBG
4 (-) binds to these residues in the anionic form,
which has an absorbance maximum at 595 nm
Benedict's reagent when heated with (blue). The free dye in solution is in the cationic
glucose or fructose reduces the blue copper (II) form, which has an absorbance maximum at 470
ion to form a positive result by the presence of nm (red). The assay is monitored at 595 nm in a
brick red solution of copper (I) oxide. Positive spectrophotometer, and thus measures the CBBG
results (red solutions) were obtained for test (see figure) complex with the protein.
tubes 1, 2 and 3.
For test tube 1 and 2 a positive result
was obtained. Test tube 1 was found to be darker
than test tube 2 because test tube 1 has a higher
concentration of glucose and fructose due to the
presence of the catalyst invertase which speed up
the reaction of sucrose. Test tube 2 does not
contain invertase and hence a slow reaction is
present which means less amounts of glucose
and fructose were formed during the time.
Test tube 3 was proven positive and the
darkest of them all because aside from the
presence of invertase, the solution already
contains glucose and fructose and thus making it
highly concentrated.
The result for test tube 4 was negative
because of the absence of sucrose, a non- One crucial part of this assay is the
reducing sugar which does not react with buffer blank. Since the assay responds non-
Benedict’s reagent linearly it is highly important to lock down the
zero point. Because this point is so important to
Warburg-Christian method the curve fit, it is highly recommended that at
Proteins exhibit a strong absorption at least two buffer blanks be performed. The
280 nm due to the absorption of the aromatic absorbance and concentrations are tabulated
rings of their intrinsic tyrosine and tryptophan below.
amino acid residues. This includes nucleic acids
that are one of the most common contaminants For Albumin:
of protein preparations. The data obtained is Protein Concentration
Test Tube # A595
(mg/mL)
tabulated below. Albumin was not analyzed due 1 0.559 0.024
to spectrometer malfunction. 2 0.884 0.0359
3 0.0734 0.004
Protein 4 0.0786 0.0013
Protein
A280 A260 A280 / A260 Concentration 5 0.0877 0.0044
Extract
(mg/mL) 6 0.093 0.0046
0.417 0.412 7 0.0643 0.0034
Casein 1.01 0.3329
1 7 8 0.0664 0.0035
9 0.0644 0.0034
By using the table of the absorbance of 10 0.0628 0.0034
11 0.0612 0.003
nucleic acid, the casein concentration can be 12 0.0619 0.0033
computed by linear regression.
For Casein:
 Protein Concentration
Test Tube # A595
(mg/mL)
7 0.0808 0.0041
8 0.0821 0.0038
9 0.0975 0.0048
10 0.1033 0.0051
11 0.1244 0.0059
12 0.1190 0.0052

The concentrations can be calculated

using Beer’s Law. Casein has a higher
absorbance reading than albumin which means
that casein has more proteins that binds with
CBBG.

CONCLUSION

The redder the solution in the

Benedict’s test, the more concentrated is the
glucose and fructose in the solution. The enzyme
invertase helps catalyzes the solution.
The Warburg-Christian method was
used to determine the concentration of Casein
which was 0.3329 mg/mL. Proteins are absorbed
at 280 nm while nucleic acid absorbs at the 260
nm region.
Coomassie brilliant blue dye reacts with
certain amino acids which affects the absorbance
reading. Absorbance is directly proportional to
concentration.

LITERATURE CITED

1. Biochemistry, Garrett and Grisham, 3rd Edition

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