ISOLATION, ACID HYDROLYSIS & COLOR REACTIONS OF HYDROLYZED MYOGLOBIN

Zarah G. Agbuya, Janmaverick D. Alarcio, Adrian R. Alvarez,

Trisha V. Aquino, Daniel O. Aracan, and Elain I. Arenas
Group 1 2B Pharmacy Biological Chemistry Laboratory
The experiment involved the isolation of myoglobin, hydrolysis of the intact protein and the qualitative reactions of
hydrolyzed myoglobin. Myoglobin was isolated from beef. Acid hydrolysis denatured the intact protein. Different
qualitative reactions were employed to test the hydrolyzed myoglobin. Only Ninhydrin test, Xanthoproteic test and
Nitroprusside test gave negative results. Biuret reaction accounted for determination of the presence of peptide
bonds, Ninhydrin reaction was employed for -amino acids. Xanthroproteic test was used to detect the side-chains of
aromatic amino acids. Millons test was employed for the determination of tyrosine residues. Hopkins-Cole tests was
employed for the determination of tryptophan residues. Sakaguchi reaction was used to detect arginine.
Nitroprusside test was used to test for sulfur containing amino acids . Fohls test was also used to know if sulfurcontaining amino acids were present. Test for amides determined the presence of amides. Pauly Test detected the
presence of nitrous acid and hydrochloric acid.

INTRODUCTION
Proteins, along with lipids and carbohydrates,
are important classes of biochemical molecules
which are essential for life. Proteins serve as the
basis for the major structural components of
tissues comprising a living organism specifically
humans and animals.
Proteins are the natural polymers of amino
acids. Amino acids are covalently linked through
peptide bonds to form linear polymers called
peptides or polypeptide chain. [1] The physical
and chemical properties which are unique to
each amino acid are the result of the structure
and chemical properties of the R group. Amino
acids have different chemical reactive groups
providing a wide range of reactivity proteins. The
reactions for individual side-chain radicals and amino and -carboxyl groups are specific both
for free amino acids and proteins. [4] Some
reactions used for identifying amino acids and
proteins in biological media, for qualitative and
quantitative analysis include Biuret, Ninhydrin,
Xanthroproteic, Millon, Hopkins-Cole, Sakaguchi,
Nitroprusside, Fohl, Test for Amides and Pauly
tests.
Myoglobin is a kind of protein found in the
muscle tissue of vertebrates. It is a singlechain globular protein of 153 or 154 amino acids,
containing a heme, which is an iron-containing
porphyrin prosthetic group in the center around
which the remaining apoprotein folds. [6]
Biologically, active proteins, like myoglobin, are
made up of polymers consisting of amino acids
linked by covalent peptide bonds. [7] These
bonds are broken when the protein undergoes
hydrolysis. In hydrolysis, the protein is subjected
to
extreme
conditions
usually
at
high
temperatures by prolonged boiling in a strong
acid or strong base or using an enzyme such
as the pancreatic protease enzyme to stimulate
the naturally occurring hydrolytic process. This
will cause denaturation of the protein meaning

that the proteins conformation is altered by the

breaking of peptide bonds. This results to a
solution containing amino acid fragments which
is then called the hydrolysate. [2]
The objective of the experiment is to test the
hydrolyzed myoglobin using different qualitative
reactions, so as to determine the chemical
affinities of the hydrolyzed protein.

EXPERIMENTAL
A. Compounds tested (or Samples used)
The compound used in this experiment varied
by group. In the isolation of myoglobin, minced
beef muscle was used. In the acid hydrolysis of
the intact protein, myoglobin was used. In the
qualitative color reactions hydrolyzed proteins,
which were myoglobin extracted from ground
beef and was hydrolyzed by acid after it was
tested with different qualitative color tests, was
used. [3]
B. Procedure
1. Isolation of Proteins: Myoglobin from
Muscle
For this type of isolation, 6.0 g of minced beef
heart and 6 mL 70% (NH4)2SO4 solution was
placed in a small beaker. It was stirred gently for
1 minute. Cheesecloth was used to express the
dark-extract. The extract was centrifuged at
13,000x g for 5 minutes. 1.5 mL of the
supernatant was transferred into another empty
centifruge tube. 0.35 g of (NH4)2SO4 crystals
were grounded to fine powder and mixed gently.
The sample was centrifuged again for 5 minutes.
The supernatant was decanted so that the
sample may be hydrolyzed. [3]
2. Acid Hydrolysis of Intact Protein
For this type of hydrolysis, 10 ml of the 6M HCl
was added to 0.5 g of the isolated myoglobin in a
hard glass test tube. The test tube was properly
labeled according to the format given. Then
cotton was placed to stopper the tube before it

was subjected to autoclaving at 15 psi for 5

hours. Alternatively, the protein sample could be
placed in a sealed container containing 6M HCl.
The whole container is then placed in a
microwave oven for about 5-30 minutes with
temperatures up to 200oC. This will vaporize the
6M HCl which will come in contact with
myoglobin
and
hydrolyse
it.
Then,
the
appearance of the mixture was noted after
autoclaving. Then, distilled water with a volume
of 10 mL was added and the mixture was
transferred into a 250-mL beaker. The mixture
was neutralized with 1M NaOH and that
neutralized mixture was used for characterization
tests and chromatography. [3]
3. Qualitative Color Reactions: Biuret Test
Eight test tubes were prepared for the
performance of the eight different test reactions.
Each test tube consisted of 0.5 mL of hydrolyzed
samples added to 1 mL of distilled water. In the
Biuret test, 20 drops of 2.5M NaOH was added to
one test tube and mixed. 3 Drops of CuSO4
solution was also added. The test was shaken
and was noted for color changes. [3]
4. Qualitative Color Reactions: Ninhydrin
Test
In Ninhydrin test, 6-10 drops of 0.1% Ninhydrin
solution was placed into the diluted samples and
heated in a water bath. The appearance of a
blue-violet coloration was noted. [3]
5.
Qualitative
Color
Reactions:
Xanthoproteic Test
In Xanthroproteic test, 10 drops of concentrated
HNO3 and 10 drops of concentrated NaOH were
added slowly and then mixed. Color changes
after each addition were observed.
6. Qualitative Color Reactions: Millons
Test
In Millons test, 5 drops of Millons reagent was
added to the diluted sample and the color
changes were noted. [3]
7. Qualitative Color Reactions: HopkinsCole test
In Hopkins-Cole test, 20 drops of Hopkins-Cole
reagent was slowly added to the samples and
was mixed well. 20 drops of H 2SO4 was slowly
added into an inclined test tube and the color
changes were noted. [3]
8. Qualitative Color Reactions: Sakaguchi
Test
In Sakaguchi test, 10 drops of 10% NaOH and
10 drops of 0.02% naphthol solution were added
to the samples and were mixed. 3 minutes was
allotted to let it stand. 3 drops of 2% NaOBr was
added, was mixed and was noted for changes.
[3]
9.
Qualitative
Color
Reactions:
Nitroprusside test

In Nitroprusside test, 0.5 mL of 3M NaOH was

added to 0.5 mL of sample. 0.25 mL of 2%
nitroprusside solution was also added and the
formation of a red solution was also noted. [3]
10. Qualitative Color Reactions: Fohls Test
In Fohls test, 5 drops of 30% NaOH and 2 drops
of 5% (CH3COO)2Pb was added to the samples in
a tube and was placed in a water bath. The
appearance was noted. [3]
11. Qualitative Color Reactions: Test for
Amides
In Test for Amides, 1 mL of 20% NaOH and 10
drops of the sample was added together in a
tube and was placed in a water bath. The test for
evolution of gas was performed by placing a
moistened red litmus paper over the mouth of
the tube and the results were noted. [3]
12. Qualitative Color Reactions: Pauly Test
In Pauly Test, the diazo reagent was prepared by
mixing 5 drops of 1% sulfanilic acid with 3 drops
5% NaNO2 solution. 5 drops of the sample and 5
drops of 10% Na2CO3 to the diazo reagent and
the appearance was noted. [3]

RESULTS AND DISCUSSION

1. Isolation of Myoglobin
After isolation, reddish brown with beef lumps
was formed.
2. Acid Hydrolysis of Intact Proteins
After hydrolysis, a clear solution was formed at
the top while turbid at the bottom with a reddish
brown color. In acidic hydrolysis, the isolated
myoglobin the 6M HCl served as the medium for
hydrolysis. In other words, it is the HCl that
serves as the catalyst for the denaturation of
myoglobin. The mixture was then subjected to
autoclaving or it could either been put into a
microwave at high temperatures. The autoclave
machine provided an environment with a
pressure of 15 psi (pound per square inch)
enough to cause denaturation of the protein if
done in a sufficient amount of time. Alternatively,
if the isolated myoglobin was put in a microwave
containing 6M of HCl and with a temperature of
200oC, it would supply the mixture a temperature
which is suitable for denaturation as well. The
acid catalyst which is the HCl will vaporize and
will eventually react with the protein, thus
hastening the hydrolysis. From a pale red turbid
solution, the hydrolysate turned into a dark
brown solution after autoclaving. Then, the
mixture was neutralized by 1M NaOH to make an
appropriate solution for the qualitative reactions.
This neutralization will cause the denaturation of
myoglobin because the acid-base interaction will
form a salt bridge that disrupts the proteins
structure.
3. Qualitative Color Reactions

After the performance of each of the

qualitative reactions, these were the results.
Ninhydrin
test,
Xanthoproteic
test
and
Nitroprusside test didnt give the desired visible
positive result, which means the absence of
cyclic a m i n o a c i d s , alpha amino acid, and
sulfur-containing alpha amino acids in the
hydrolyzed proteins, while Biuret test, Millons
test, Hopkins-Cole test, Sakaguchi test, Fohls
test, Test for Amide and Pauly test.
Biuret test is used to identify the presence of
protein residues and is usually done in food
microbiology. Ninhydrin test indicates the
presence of alpha amino acids in proteins.
Xanthroproteic reaction is specific for cyclic
amino acids such as phenylalanine,
t y r o s i n e , tryptophan and histidine. Aromatic
amino acids react with nitric acid, which yield
yellow nitro-compounds, which changes color
to orange i n a l k a l i n e m e d i u m o w i n g t o
t h e f o r m a t i o n o f salt positive for the basic
hydrolysis.
Since
histidine
and
t r y p t o p h a n a r e a r o m a t i c amino acids,
Xanthroproteic reaction yielded a positive
result for the intact protein but not for the
hydrolysate caused probably by the errors
or loss of protein during hydrolyzation process.
Millons reagent is used for the determination of
tyrosine content in proteins. It i s c o m p o s e d
o f s a l t s o f m e r c u r y d i s s o l v e d i n nitric
acid. Tyrosine, which is both free and
constitutive of proteins, reacts with reagent
and produces a purple-red salt of mercury.
Hopkins-Cole are used to identify tryptophan
residues. Sakaguchi reaction is typical for
arginine only. Arginine reacts with naphtol and produces a red colored derivative.
The presence o f a r g i n i n e i s p o s i t i v e f o r
both
the
intact
and
hydrolyzed
myoglobin
and
supported
by
the
presence of arginine in the paper
chromatography
of
hydrolyzed
p r o t e i n . Nitroprusside test is a specific test for
sulfur containing amino acids such as cysteine,
cysteine and methionine. Fohls test is also used
for the determination of S-containing amino
acids. Heating of myoglobin solution in an
alkaline medium leads to the formation of
sodium sulfide, if the protein contained
sulfur amino acids such as cysteine,
cystine, or methionine. In further reaction
of sodium sulfide with lead acetate a dark
brown colored precipitate is formed. Test for
amides determines the presence of amides.
Pauly Test determines the presence of nitrous
acid and hydrochloric acid, which results to the
formation of highly colored azo-compounds. This
is all represented in Table 1. [5]

Table 1: Qualitative Reactions Results

Color Reaction
Hydrolyzed
Protein
(ACIDIC)
Biuret
Ninhydrin
Xanthroproteic
Millons
Hopkins-Cole
Sakaguchi
Nitroprusside
Fohls
Test for Amide
Pauly

Formation of
light blue
solution (clear)
Formation of a
clear violet
solution
Formation of a
clear solution
Formation of a
slightly cloudy
solution
Formation of a
clear solution
Formation of a
yellow solution
Formation of a
clear yellow
solution
Formation of a
clear solution
Red Litmus
turned blue
Formation of
orange solution

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