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 quantitatively determine the protein concentration
Proteins are a long chain-like molecule that is of a given sample using commonly used and
made up of small units known as amino acids, microscale methods of total protein analysis.
linked by covalent peptide bonds. [1] It is the most
abundant biomolecules inside the human body and EXPERIMENTAL
is very essential for life. It plays critical roles such A. Test Compound/s (or Sample/s) used
as supplying for body structures as keratin, Intact protein and hydrolyzed samples (basic
providing frameworks for connective tissues, and enzymatic) of Myoglobin.
maintaining different physiological process as
enzymes and transporting materials throughout B. Procedure
the body as myoglobin, a protein that is very
important for muscle tissues. 1. Isolation of Protein
Myoglobin is a single-chain globular protein that The protein isolated from the minced beef was
consists of 153 amino acids and a heme group (an myoglobin. Myoglobin was extracted by using
iron-containing porphyrin). It is a small oxygen- ammonium sulfate precipitation on the buffered
binding protein found in muscle cells. Its functions muscle extract.
primarily in storing oxygen and facilitating oxygen 6 g of the meat was mixed together with 6 ml of
diffusion in muscle tissue. [2] Myoglobin are active 70% ammonium sulfate and then stirred for 1
proteins made up of polymers of amino acids minute. The meat was then put in a cheesecloth to
joined by covalent bonds. These bonds are broken separate the dark-red extract from the meat into
through the process of hydrolysis. In hydrolysis, another beaker. The following residual extract was
proteins are exposed to extreme conditions at high then centrifuged at 13,000 x g for 5 minutes. After
temperature using acids, alkali and proteolytic centrifugation, 1.5 mL of the supernatant liquid
enzymes. This will eventually trigger the was then transferred into another centrifuge tube
denaturation of protein which means the and then 0.3-0.35 g of ammonium sulfate crystals
conformation of protein is altered by the breaking was added. The substance was centrifuged again
of peptide bonds which resulted into a solution for 5 minutes then the supernatant was decanted
containing fragments of amino acids which is off.
called hydrolysate.
The experiment has the following objectives: 1) 2. Acid Hydrolysis of Intact Protein
to isolate the protein myoglobin from minced beef The protein is treated with acid enzymes to
using the principle salt-induced precipitation. 2) to obtain information about its composition. 5 mL of
perform qualitative test on amino acids in intact 6 M hydrochloric acid is added to 0.5 g of the
and hydrolyzed myoglobin and explain the isolated protein (myoglobin) in a hard glass test
principles involved in each test. 3) to execute tube. The test tube is then covered with cotton and
alkaline and enzymatic hydrolysis on the isolated submitted to the instructor to be autoclaved at 15
myoglobin and enumerate the advantages and psi for 5 hours. 10 mL of distilled water is added
disadvantages of using each type of hydrolysis. 4) after autoclaving and was then transferred into a
to determine the amino acid components of 250-mL beaker. Neutralization of the substance
myoglobin using thin-layer chromatography. 5) to was done by adding 1 M sodium hydroxide.
 Nitroprusside Test
3. Qualitative Color Reactions .5 mL of 3M NaOH and .25 mL of 2%
For the 10 qualitative color tests, 30 test tubes nitroprusside solution were added to each sample,
were collected. Every test was allotted three test respectively. Formation of red solution was noted
tubes, one containing .5 g of intact protein in 1 mL in each sample.
distilled water, another one containing .5 mL of
enzymatic hydrolysate and the last one containing Folh’s Test
.5 ml of alkaline hydrolysate. Each sample was added 5 drops of 30% NaOH
and 2 drops of 5% Pb(CH3COO)2, respectively. The
Biuret Test three test tubes were then placed into a boiling
20 drops of 2.5 M NaOH were added to each water bath and the appearance of dark (black or
sample. The test tubes were mixed well and then brown) sediments were noted in each sample.
to each one 2-3 drops of 0.1 M CuSO4 solution
were added. The test tubes were shaken, and the Test for Amides
color of the solution was recorded. 1 mL of 20% NaOH were added to every 10
drops of each sample. The test tubes were then
Ninhydrin Test placed in a boiling water bath. A red litmus paper
6-10 drops of 0.1% ninhydrin solution were was placed over the mouth of each test tube then
placed in the three samples. The test tubes were the results were noted.
then heated in a boiling water bath and the
appearance of blue-violet coloration was recorded. Pauly’s test
The diazo reagent was prepared first by mixing
Xanthoproteic Test 3-5 drops of 1% sulfanilic acid with 3 drops of 5%
Ten drops of conc. HNO3 were slowly added to NaNO2 solution. 5 drops of each sample and 3-5
each sample then the color of the solution was drops of 10% Na2CO3 were then added to the diazo
noted. After that 10 drops of conc. NaOH were reagent made. The appearance of a red coloration
slowly added as well in each sample then each was recorded.
were mixed well, and the color of the solution was
recorded. 4. Separation and Identification of Amino
Acid by Paper Chromatography
Millon’s Test
5 drops of Millon’s reagent were added to the A 12 x 15 TLC plate was prepared, and a
three samples then the change in color of each 1.5 cm margin from the bottom of the longer end
solution was noted. of the plate was drawn light lightly with a pencil.
Six equidistant points were marked, 4 amino acid
Hopkins-Cole Test standards and 2 hydrolysate samples (Alkaline,
In a gradual manner, 20 drops of Hopkins-Cole Enzymatic). Using a capillary tube, the amino acid
reagents were added to the samples. Each test standards were applied 3 times while the
tubes were mixed well and were inclined first hydrolysed samples were applied 5 times, and the
before slowly adding along the side, 20 drops of samples were air-dried in between applications.
conc. H2SO4. The mixtures were not shaken and The plate was rolled into a cylinder without
the color at the interface was noted. overlapping and then stapled. Then, it was placed
inside a pre-equilibrated chamber, wherein the
Sakaguchi Test level of solvent was below the origin. The chamber
10 drops of 10% NaOH and 10 drops of 0.2% was covered and allowed the solvent to ascend
naphthol solution were added to each test tube undisturbed. Once the solvent has reached 2 cm
containing the samples, respectively. The test from the other end, the plate was removed and
tubes were mixed and allowed to stand for 3 the solvent front was immediately marked lightly
minutes. Then 3 drops of 2% NaOBr were added. with a pencil. The chromatogram was air-died and
The three test tubes were mixed again, and the a 1% ninhydrin reagent was applied to it. Next,
color produced was recorded. 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]