Isolation and Characterization of Myoglobin

Troy Culder C. Sze, Glenn L. Tan, Andrea P. Ventura,

Miguel Alfred B. Vicente and Precious Joy H. Vistan
Group 8 2D Medical Technology Biochemistry Laboratory

ABSTRACT

A 250 mL Tertiary phosphate buffer solution with a 12.00 pH with 6.0 molar concentration was prepared by using 3.38
NaOH and 33.5 HPO42-. The pH of the buffer was adjusted to the desired pH value using 6M NaOH, while being monitored
by a pH meter. Colorimetric determination was then performed to determine the range of pH of the buffer after color
change.
INTRODUCTION
Proteins are a long chain-like molecule that is
made up of small units known as amino acids,
linked by covalent peptide bonds. [1] It is the most
abundant biomolecules inside the human body and
is very essential for life. It plays critical roles such
as supplying for body structures as keratin,
providing frameworks for connective tissues,
maintaining different physiological process as
enzymes and transporting materials throughout
the body as myoglobin, a protein that is very
important for muscle tissues.
Myoglobin is a single-chain globular protein that
consists of 153 amino acids and a heme group (an
iron-containing porphyrin). It is a small oxygen-
binding protein found in muscle cells. Its functions
primarily in storing oxygen and facilitating oxygen
diffusion in muscle tissue. [2] Myoglobin are active
proteins made up of polymers of amino acids
joined by covalent bonds. These bonds are broken
through the process of hydrolysis. In hydrolysis,
proteins are exposed to extreme conditions at high
temperature using acids, alkali and proteolytic
enzymes. This will eventually trigger the
denaturation of protein which means the
conformation of protein is altered by the breaking
of peptide bonds which resulted into a solution
containing fragments of amino acids which is
called hydrolysate.
The experiment has the following objectives: 1)
to isolate the protein myoglobin from minced beef
using the principle salt-induced precipitation. 2) to
perform qualitative test on amino acids in intact
and hydrolyzed myoglobin and explain the
principles involved in each test. 3) to execute
alkaline and enzymatic hydrolysis on the isolated
myoglobin and enumerate the advantages and
disadvantages of using each type of hydrolysis. 4)
to determine the amino acid components of
myoglobin using thin-layer chromatography. 5) to
quantitatively determine the protein concentration
of a given sample using commonly used and
microscale methods of total protein analysis.
EXPERIMENTAL
A. Test Compound/s (or Sample/s) used
Intact protein and hydrolyzed samples (basic
and enzymatic) of Myoglobin.
B. Procedure
1. Isolation of Protein
The protein isolated from the minced beef was
myoglobin. Myoglobin was extracted by using
ammonium sulfate precipitation on the buffered
muscle extract.
6 g of the meat was mixed together with 6 ml of
70% ammonium sulfate and then stirred for 1
minute. The meat was then put in a cheesecloth to
separate the dark-red extract from the meat into
another beaker. The following residual extract was
then centrifuged at 13,000 x g for 5 minutes. After
centrifugation, 1.5 mL of the supernatant liquid
was then transferred into another centrifuge tube
and then 0.3-0.35 g of ammonium sulfate crystals
was added. The substance was centrifuged again
for 5 minutes then the supernatant was decanted
off.
2. Acid Hydrolysis of Intact Protein
The protein is treated with acid enzymes to
obtain information about its composition. 5 mL of
6 M hydrochloric acid is added to 0.5 g of the
isolated protein (myoglobin) in a hard glass test
tube. The test tube is then covered with cotton and
submitted to the instructor to be autoclaved at 15
psi for 5 hours. 10 mL of distilled water is added
after autoclaving and was then transferred into a
250-mL beaker. Neutralization of the substance
was done by adding 1 M sodium hydroxide.

3. Qualitative Color Reactions
For the 10 qualitative color tests, 30 test tubes
were collected. Every test was allotted three test
tubes, one containing .5 g of intact protein in 1 mL
distilled water, another one containing .5 mL of
enzymatic hydrolysate and the last one containing
.5 ml of alkaline hydrolysate.
Biuret Test
20 drops of 2.5 M NaOH were added to each
sample. The test tubes were mixed well and then
to each one 2-3 drops of 0.1 M CuSO4 solution
were added. The test tubes were shaken, and the
color of the solution was recorded.
Ninhydrin Test
6-10 drops of 0.1% ninhydrin solution were
placed in the three samples. The test tubes were
then heated in a boiling water bath and the
appearance of blue-violet coloration was recorded.
Xanthoproteic Test
Ten drops of conc. HNO3 were slowly added to
each sample then the color of the solution was
noted. After that 10 drops of conc. NaOH were
slowly added as well in each sample then each
were mixed well, and the color of the solution was
recorded.
Millon’s Test
5 drops of Millon’s reagent were added to the
three samples then the change in color of each
solution was noted.
Hopkins-Cole Test
In a gradual manner, 20 drops of Hopkins-Cole
reagents were added to the samples. Each test
tubes were mixed well and were inclined first
before slowly adding along the side, 20 drops of
conc. H2SO4. The mixtures were not shaken and
the color at the interface was noted.
Sakaguchi Test
10 drops of 10% NaOH and 10 drops of 0.2%
naphthol solution were added to each test tube
containing the samples, respectively. The test
tubes were mixed and allowed to stand for 3
minutes. Then 3 drops of 2% NaOBr were added.
The three test tubes were mixed again, and the
color produced was recorded.
Nitroprusside Test
.5 mL of 3M NaOH and .25 mL of 2%
nitroprusside solution were added to each sample,
respectively. Formation of red solution was noted
in each sample.
Folh’s Test
Each sample was added 5 drops of 30% NaOH
and 2 drops of 5% Pb(CH3COO)2, respectively. The
three test tubes were then placed into a boiling
water bath and the appearance of dark (black or
brown) sediments were noted in each sample.
Test for Amides
1 mL of 20% NaOH were added to every 10
drops of each sample. The test tubes were then
placed in a boiling water bath. A red litmus paper
was placed over the mouth of each test tube then
the results were noted.
Pauly’s test
The diazo reagent was prepared first by mixing
3-5 drops of 1% sulfanilic acid with 3 drops of 5%
NaNO2 solution. 5 drops of each sample and 3-5
drops of 10% Na2CO3 were then added to the diazo
reagent made. The appearance of a red coloration
was recorded.
4. Separation and Identification of Amino
Acid by Paper Chromatography
A 12 x 15 TLC plate was prepared, and a
1.5 cm margin from the bottom of the longer end
of the plate was drawn light lightly with a pencil.
Six equidistant points were marked, 4 amino acid
standards and 2 hydrolysate samples (Alkaline,
Enzymatic). Using a capillary tube, the amino acid
standards were applied 3 times while the
hydrolysed samples were applied 5 times, and the
samples were air-dried in between applications.
The plate was rolled into a cylinder without
overlapping and then stapled. Then, it was placed
inside a pre-equilibrated chamber, wherein the
level of solvent was below the origin. The chamber
was covered and allowed the solvent to ascend
undisturbed. Once the solvent has reached 2 cm
from the other end, the plate was removed and
the solvent front was immediately marked lightly
with a pencil. The chromatogram was air-died and
a 1% ninhydrin reagent was applied to it. Next,
the chromatogram was placed on top of the hot
plate and observed for the amino acids that
appeared as blue, purple and yellow spots on the Fohl’s no no No change/
chromatogram. black/br black/br colorless
own own
RESULTS AND DISCUSSION sedimen sedimen
ts ts
1.Isolation of Protein
Test for Red to Red to Red to blue
Amide blue blue litmus paper
2. Qualitative Color Reactions
litmus litmus
paper paper

Pauly’s Colorles Yellow Red

Color Intact Hydrolyzed Protein s
Reaction Protein
Table 1. Qualitative Color Reactions
Basic Enzymatic

Biuret Purple Light Blue

blue

Ninhydrin Light Colorles Light violet

violet s

Xanthoprot Light Colorles Light yellow

eic yellow s

Figure 1. Qualitative Color Reactions on

Enzymatic Hydrolyzed Myoglobin.
Millon’s Colorles Colorles colorless
s s

Hopkins- No No violet No violet

Cole violet ring ring
ring

Sakaguchi red colorless

colorles
s

Nitroprussi Yellow Yellow yellow

de
REFERENCES

[1]http://www.sciencekids.co.nz/sciencefacts/foo
d/proteins.html
[2]
https://en.wikibooks.org/wiki/Structural_Bioche
mistry/Myoglobin#Myoglobin's_further_importanc
e
[3]