Isolation and Characterization of Proteins

Proteins, from the Greek proteios, meaning first, are a class of organic compounds which are present in and vital to
every living cell. In the form of skin, hair, callus, cartilage, muscles, tendons and ligaments, proteins hold together,
protect, and provide structure to the body of a multi-celled organism. In the form of enzymes, hormones, antibodies,
and globulins, they catalyze, regulate, and protect the body chemistry. In the form of hemoglobin, myoglobin and
various lipoproteins, they affect the transport of oxygen and other substances within an organism. Myoglobin, a
protein found in the muscle cells of animals functions as an oxygen-storage unit, providing oxygen to the working
muscles. Diving mammals such as seals and whales are able to remain submerged for long periods because they
have greater amounts of myoglobin in their muscles than other animals do. Myoglobin, the intact protein from beef
was extracted and hydrolyzed by acid and base and subjected to different qualitative color tests.

Experimental
A. Compounds tested
1. Isolating intact protein
Beef
70% (NH4)2SO4
2. Hydrolysis of intact proteins
Myoglobin
6 M HCl
4 M NaOH
3. Qualitative Color Reactions
Myoglobin
Hydrolyzed acid
Hydrolyzed base
4. Separation and identification of amino
acids by thin-layer chromatography
Amino acid standards
Hydrolyzed samples
5. Protein assay using the Bradford
method
Bovine serum albumin (BSA)
B. Procedure
1. Isolation of protein (Myoglobin)
In a beaker, 6.0 g of minced beef and 6 mL
70% (NH4)2SO4solution was placed.
Afterwards, the mixture was gently stirred
for 1 minute to release the myoglobin. The
dark red extracts were then pressed into a
new beaker using cheesecloth. The extract
was then centrifuged at 13,000x g for 5
minutes. Then, 1.5 mL of the supernatant
was transferred into another empty
centrifuge tube. (NH4)2SO4 Crystals with
weight of ~0.30-0.35 g ground to fine
powder were then added and mixed gently
until the solid dissolved. The sample was

centrifuged again for 5 minutes and the

supernatant was decanted off. The
appearance of the purified myoglobin
residue was described afterwards.
2. Hydrolysis of Intact Proteins
a. Acid hydrolysis of intact protein
In a hard glass test tube, 5 mL of 6 M HCl
was added to 0.5 g isolated protein. The
test tube was then labeled and cotton was
placed as a stopper. Afterwards, it was
submitted to the instructor for autoclaving
(15 psi for 5 hours). The appearance of the
mixture was then noted. Then, 10 mL of
distilled water is then added. The mixture
was then transferred into a 250-mL beaker.
The mixture was then neutralized with 1 M
NaOH. The neutralized mixture was used as
a sample for characterization tests and
chromatography
b. Alkaline hydrolysis of intact protein
In a hard glass test tube, 10 mL of 4 M
NaOH was added to 0.5 g isolated protein.
A stopper is then placed to the stopped and
the test tube was then labeled and
submitted to the instructor. The appearance
was noted after autoclaving. 10 mL of
distilled water was then added and the
mixture was then transferred into a 250 mL
beaker. The mixture is then neutralized with
1 M HCl. The neutralized mixture was used
as a sample for characterization tests and
chromatography.
3. Qualitative Color Reactions
For each test, separate test tubes were
prepared for each of the color tests. Each
test tube contained an intact protein solution
(0.5 g of the protein in 1 mL distilled water)
and 0.5 mL of hydrolyzed sample. For the

Biuret test, 20 drops of 2.5 M NaOH was

added to the samples and mixed. 203 drops
of 0.1 M CuSO4 solution was then added
and the test tube was shaken and the color
of the solution was noted. For Ninhydrin
test, 6-10 drops of 0.1% ninhydrin solution
was placed into the diluted samples. The
test tube was heated in a boiling water bath.
For Xanthoproteic test, 10 drops of conc.
HNO3 was slowly added to the diluted
samples, mixed, and the color of the
solution was noted. Afterwards, 10 drops of
conc. NaOH was slowly added, mixed, and
the color of the solution was noted again.
For Millons test, 5 drops of Millons reagent
was added to the diluted samples and the
color change was noted. For Hopkins-Cole
test, 20 drops of Hopkins-Cole reagent was
slowly added to the samples and mixed
well. The test tube was then inclined and 20
drops of conc. H2SO4 were added slowly
along the side of the test tube. The mixture
was not shaken and the color at the
interface was noted. For Sakaguchi test, 10
drops of 10% NaOH and 10 drops of 0.02%
naphthol solution was added to the
samples, mixed, and allowed to stand for
3minutes. Afterwards, 3 drops of 2% NaOBr
was added, mixed, and the color produced
was noted. For Nitroprusside test, 0.5 mL of
3 M NaOH was added to 0.5 mL of the
sample. Then, 0.25 mL 2% nitroprusside
solution was added. For Fohls test, 5 drops
30% NaOH and 2 drops 5% (CH3COO)2Pb
was added to the samples and the tube was
placed in a boiling water bath. For Test for
Amides, 1 mL of 20% NaOH was added to
10 drops of the sample and the test tube
was placed in a boiling water bath.
Afterwards, there was a test for the
evolution of gas during heating by placing a
moistened red litmus paper over the mouth
of the test tube. The result was then noted.
For Pauly test, the diazo reagent was

prepared by mixing 3-5 drops 1% sulfanilic

acid with 3 drops 5% NaNO2 solution.
Afterwards, 5 drops of the sample and 3-5
drops 10% Na2CO3 was added to the diazo
reagent.
4. Separation and identification of amino
acids by thin-layer chromatography
A 1.5 cm margin from one edge of the paper
was measured and drawn lightly with a
pencil on the prepared 12 x 20 cm
Whatman filter paper. Ten equidistant points
were labeled on the line for application of
the samples. The samples were air-dried
between applications by a capillary tube.
The paper was then rolled into a cylinder
without overlapping and then stapled. It was
then placed on the pre-equilibrated chamber
and then covered for the solvent to ascend.
When the solvent reached at least 2 cm
from the other end, the paper was removed
and the solvent font was immediately
marked. The paper was air-dried and
sprayed lightly with ninhydrin reagent. Then
it was oven-dried and observed for the
amino acids that appeared as blue, purple
and yellow spots on the paper. The spots
were encircled and computed for the Rf
values.
5. Protein assay using the Bradford
method
Results and Discussions:
Resources:
http://www.britannica.com/EBchecked/topi
c/400480/myoglobin

https://www2.chemistry.msu.edu/faculty/re
usch/virttxtjml/proteins.htm