ISOLATION AND HYDROLYSIS OF INTACT PROTEIN: MYOGLOBIN

Erika C. Rabara, Harvey Mher M. Rarang, Maika L. Regala, Chelejan Mhare U. Regino, Erik
Kristian Victor B. Sabio, Mica Gienela A. Sanchez, Arianne Nicole Denise T. Yoro
Group 8
2D Medical Technology
Biochemistry Laboratory

ABSTRACT
Myoglobin is the primary oxygen-carrying pigment of muscle tissues. It contains hemes, pigments responsible for the
color of red meat. The color that meat takes is partly determined by the degree of oxidation of the myoglobin. This
portion of experiment refers to the denaturation of intact myoglobin through hydrolysis. The pre-isolated myoglobin
from meat sample was subjected to basic hydrolysis to alter its native conformation and thus making a suitable
hydrolysate for qualitative and quantitative characterization. NaOH was used as a medium in basic hydrolysis and HCl
was used to neutralize the mixture while in acid hydrolysis, HCl was the medium used and NaOH was the neutralizing
agent. In enzymatic hydrolysis, however, a protease solution was used as a medium and the mixture was incubated in
a water bath for a specific temperature. It has been concluded that in hydrolysing proteins, basic and acid hydrolysis
is more effective and efficient than enzymatic hydrolysis because both acid and basic hydrolysis undergoes complete
hydrolysis while the latter undergoes incomplete hydrolysis,

INTRODUCTION
Myoglobin
is
a single-chain globular
protein of 153 or 154 amino acids, containing
a heme
which
is
an
ironcontaining porphyrin prosthetic
group in
the
center
around
which
the
remaining apoprotein folds. Biologically,
active
proteins, like myoglobin, are made up of
polymers consisting of amino acids linked by
covalent peptide bonds. 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. Denaturation alters
protein function, demonstrating a relationship
between structure and function. Hydrolysis of
protein and analysis of products are done to
obtain information about their compositions. The
three most common types of hydrolysis are aid,
basic and enzymatic hydrolysis. Acid hydrolysis
implies
a
chemical
mechanism
of hydrolysis catalyzed
by
a Brnsted or Arrhenius acid. By contrast, it does
not usually imply hydrolysis by direct electrophilic
attackas may originate from a Lewis acid.
Nevertheless, the type of hydrolysis carried out
with a basic medium is termed basic hydrolysis.
Lastly,
an
enzymatic
hydrolysis
is
by
the addition of specific enzymes called proteolytic
enzymes or simply proteases which refer to a

group of enzymes each to hydrolyse specific

peptide bonds of proteins. [1]

Figure 1. Structure of Myoglobin

This portion of the experiment aims to
isolate myoglobin from raw beef steak by saltinduced precipitation; and perform acid, alkaline,
and enzymatic hydrolysis on the isolated proteins
and enumerate the advantage and disadvantages
of each type of hydrolysis.

EXPERIMENTAL
A. Compounds tested (or samples used)
Buffered muscle extract and 70% bufferdiluted (NH4)2SO4 solution form crystals was used
for the isolation of myoglobin.
The reagent used for alkaline hydrolysis
was 4M NaOH; and 1M HCl was used for

neutralization purposes. Blue litmus paper was

also used to test the pH of the solution.

B. Procedure
1. Isolation of Proteins
The mixture of 20 g minced beef steak and 20
ml 70% (NH4)2SO4 were stirred for one minute to
release he myoglobin. The dark-red extract was
then expressed in the new beaker using
cheesecloth. The extract was centrifuged for 10
minutes instead of five because of the inaccuracy
of the old centrifuge. One and a half ml of
supernatant was used to dissolve 0.35 g of
(NH4)2SO4 powder.

process, the intact protein appeared to have a

dark-red turbid solution. This intact myoglobin
was subjected to hydrolysis basically to break
down the protein into smaller amino acid
fragments which will make the qualitative and
quantitative determination of its components
easier.

2. Hydrolysis of Intact Protein

a. Basic Hydrolysis of Myoglobin
Ten millilitres of 4M NaOH was placed in a
hard glass test tube containing 0.5 g of isolated
myoglobin. The mixture was submitted with a
cotton as stopper and was subjected to
autoclaving at 15 psi for five hours. The
appearance of mixture was noted. Then, 10 ml of
distilled H2O was added to the mixture and was
transferred into a 250-ml beaker. It was then
neutralized with a 1M HCl.
b. Acid Hydrolysis of Myoglobin
Five millilitres of 6M HCl was placed in a
hard glass test tube containing 0.5 g of isolated
myoglobin. The mixture was submitted with a
cotton as stopper and was subjected to
autoclaving at 15 psi for five hours. The
appearance of mixture was noted. Then, 10 ml of
distilled H2O was added to the mixture and was
transferred into a 250-ml beaker. It was then
neutralized with a 1M NaOH.
c. Enzymatic Hydrolysis
A 1g/100 mL distilled water protein mixture
was prepared. A volume of 10 mL of the protein
mixture and 10 mL of the saturated protease was
mixed. Alternatively, a 0.050 g of protease may
be added directly to 50 mL protein mixture.
Then, a 10mL 0.1 M phosphate buffer with a pH
of 7.5 was added. The tube was incubated in a
water bath maintained at 35-40 oC for about an
hour. Again, alternatively, digestion may be
carried out overnight at room temperature.

RESULTS AND DISCUSSION

1. Isolation of Proteins
Before performing hydrolysis and qualitative
color reactions, the protein, myoglobin was first
isolated from a raw beef steak. After the isolation

Figure 2. Intact myoglobin

2. Hydrolysis of Intact Protein
In basic hydrolysis, the 4M NaOH served as
the medium for hydrolysis. In other words, it is
the base that serves as the catalyst for the
denaturation of myoglobin. The mixture was then
subjected to autoclaving. The autoclave machine
provides an environment with a pressure of 15
psi, enough to cause denaturation of the protein
if done in a sufficient amount of time. The base
catalyst will vaporize and will eventually react
with the protein, thus hastening the hydrolysis.
From a dark-red turbid solution, the hydrosylate
appeared to be a colorless solution after. Then,
the mixture was neutralized by 1M HCl to make
an appropriate solution for the quantitative and
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. In protein
denaturation,
the
salt
formed
in
the
neutralization will alter the conformation of the
protein by further straitening the protein chain.
The advantage of basic hydrolysis is the
tryptophan will be stable. The downside of this
type of hydrolysis would be the racemization of
all amino acids. Arginine will be decomposed to
ornithine and urea and a partial destruction of
the nucleophilic amino acids such as cysteine,
serine, and threonine. The hydrolysis of
asparagine to aspartic acid; and glutamine to
glutamic acid will occur.
The acid hydrolysis procedure is almost the
same as the basic hydrolysis. The isolated
myoglobin was subjected to autoclaving in an

environment with a pressure of 15 psi. The only

difference is the medium used in acid hydrolysis
which is 6M HCl and neutralized by a base. The
neutralization process disrupts the native
conformation of the protein. The advantage of
acid hydrolysis is there will be little to no
racemization at all but tryptophan will be
decomposed into humin and there will be a
partial destruction of nucleophilic amino acids
such as cysteine, serine, and threonine. The
hydrolysis of asparagine to aspartic acid; and
glutamine to glutamic acid will also occur in acid
hydrolysis.
Another type of hydrolysis was not done in
myoglobin
in
the
experiment.
Enzymatic
hydrolysis uses proteolytic enzymes to cleave
only peptide bonds at specific sites. Its
advantage is that amino acids will not be affected
but this type of hydrolysis requires certain
temperature and pH to achieve optimum activity.
The enzymatic hydrolysate does not need
to be neutralized since it is neither basic nor
acidic, thus, is not susceptible to other reactions
after it cools.

REFERENCES
[1] Biochemistry of muscle, muscle contraction.
Biochemistry of connective tissue. (2013).

Retrieved March 26, 2016, from

http://intranet.tdmu.edu.ua/data/kafedra/interna
l/chemistry/lectures_stud/en/med/lik/ptn/2/20.%
20Biochemistry%20of%20muscle.html
[2] Enzymatic Hydrolysis of Food Protein for
Amino Acid
Retrieved March 26, 2016, from
http://pubs.acs.org/doi/abs/10.1021/jf60204a04
3?journalCode=jafcau

[3] Campbell, M. K., (2012./2009) Biochemistry.

China: Brooks/ Cole, Cengage Learning