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
Proteins are a long chain-like molecule that is made up of small units known as amino acids, linked by
covalent peptide bonds. These bonds can be broken down by hydrolysis. The experiment involved the
isolation of myoglobin, hydrolysis of the intact protein, and enzymatic hydrolysis of the intact protein.
Ammonium sulfate precipitation was performed to isolate myoglobin; Alkaline hydrolysis was performed by
addition of sodium hydroxide; Enzymatic hydrolysis was performed by addition of proteolytic enzymes. The
alkaline hydrolysis yielded a light-pink colored hydrolysate while the enzymatic hydrolysis yielded a ____. Commented [1]: pakilagay color result ng enzymatic
INTRODUCTION the enzymatic hydrolysis of the assigned protein
Protein isolation is a process where a single type (myoglobin).
of protein from a complex mixture. This is done to
characterize the protein’s solubility, acid-base EXPERIMENTAL
property, function structure, and interactions. A. Test Compound/s (or Sample/s) used
Separation of proteins from the complex mixture Intact protein and hydrolyzed samples (basic
would depend on their size, shape, charge, and enzymatic) of Myoglobin.
hydrophobicity, and physiochemical properties.
Some of the methods that are commonly. used are B. Procedure
heat denaturation, isoelectric precipitation, 1. Isolation of Protein
solubilization, salt-induced. precipitation, For isolation, 6 g of the meat was mixed
chromatography, and ultra-centrifugation. together with 6 ml of 70% ammonium sulfate and
Biologically, myoglobin is an active protein; then stirred for 1 minute. The meat was then put
Active proteins are made up of polymers in a cheesecloth to separate the dark-red extract
consisting of amino acids linked by covalent from the meat into another beaker. The following
peptide bonds. These bonds are then broken down residual extract was then centrifuged at 13,000 x
by hydrolysis. in hydrolysis, the protein is g for 5 minutes. After centrifugation, 1.5 mL of the
subjected to extreme conditions usually at high supernatant liquid was then transferred into
temperatures by prolonged boiling in a strong acid another centrifuge tube and then 0.3-0.35 g of
or strong base— in the case of our experiment, a ammonium sulfate crystals was added. The
strong base was used (NaOH). The base is substance was centrifuged again for 5 minutes
vaporized when heated and then comes into then the supernatant was decanted off.
contact with the protein and then hydrolyses it.
Polypeptides of a protein can unwind either 2. Alkaline Hydrolysis of Intact Protein
through exposing it with different chemicals or The protein is treated with basic enzymes to
utilizing enzymes. Naturally occurring enzymes obtain information about its composition. 10 mL of
that catalyze the hydrolytic cleavage of peptide 4 M sodium hydroxide is added to 0.5 g of the
bonds and are used in the process of enzymatic isolated protein (myoglobin) in a hard glass test
hydrolysis are called proteases. The product of the tube. The test tube is then covered with cotton and
enzymatic hydrolysis is used for a different submitted to the instructor to be autoclaved at 15
experiment. An example of such tests includes the psi for 5 hours. 10 mL of distilled water is added
determination and analysis of the amino acid after autoclaving and was then transferred into a
composition of protein. 250-mL beaker. Neutralization of the substance
The objectives of this experiment is to isolate was done by adding 1 M hydrochloric acid.
the protein myoglobin from minced beef using the
principle of salt-induced precipitation, perform 3. Enzymatic Hydrolysis of Intact Protein
alkaline hydrolysis, and to obtain the product from
 To produce the needed product for enzymatic
hydrolysis, the isolated protein was mixed with
water to produce a distilled water protein mixture
with a ratio of 1 g: 100 ml. The 10 ml of protease
solution is added and mixed with 10 ml of the
protein mixture. 10 mL 0.1 M phosphate buffer
with a pH of 7.5 was added afterward. The
resulting solution was placed in a water bath with
a temperature range of 35°C to 40°C for 60
minutes. Subsequently, the mixture was cooled Figure 1. Isolated myoglobin by ammonium
before using it for different experiments. Glenn sulfate precipitation
ano na color nung result ng enzymatic? Commented [2]: oi
2. Alkaline Hydrolysis of Protein
RESULTS AND DISCUSSION Alkaline hydrolysis is a process where complex
1. Isolation of Protein molecules are broken down into their constituent
After removing the centrifuge tube from the building blocks by addition of water (H2O), H+,
centrifuge, there was no precipitated protein pellet and OH- between the atoms of the bonds that
found in the tube, the supernatant liquid of the hold those building blocks together.
extract was used instead for the following By hydrolysing proteins, you break it
experiments. down into its component amino acids. In alkaline
Supposedly, the pellets found at the bottom of hydrolysis, some amino acids are destroyed,
the tube would be the myoglobin acquired from namely arginine, asparagine, glutamine, and
the minced beef. The pellet is formed due to the serine; alkaline hydrolysis is used in
precipitation with ammonium sulfate. Precipitation determination of tryptophan. sodium hydroxide is
the preferred alkali solutions because of the
occurs by activity of hydrophobic interactions
between protein molecules which causes instability of concentrated (50%) stock solutions
aggregation then eventually, precipitation. Since of NaOH solutions. Autoclaving was also
ammonium sulfate does not denature the protein, performed to accelerate the hydrolysis of the
but rather only reduces it’s solubility, it is possible protein since heat itself is a denaturant.
to store the precipitated protein pellets in the salt The acquired sample after autoclaving
solution to inhibit microbial growth or protease resulted in a light-pink color solution/hydrolysate
activity. which was then neutralized with 1 M hydrochloric
Solubility of globular proteins increases upon the acid to balance out the added sodium hydroxide
addition of salt, an effect termed salting-in. Salts in the solution.
that reduce the solubility of proteins also tend to
enhance the stability of the native conformation,
hence salting-in ions are usually denaturants.
when salt is added to the solution, the surface
tension of water increases, resulting in increased
hydrophobic interaction between protein and
water. The protein then decreases its own surface
area to minimize contact with the solvent (folding)
and then self-association leads to precipitation.

Figure 2. Myoglobin hydrolysate by alkaline

hydrolysis

3. Enzymatic Hydrolysis of Intact Protein

 The product obtained from the enzymatic
hydrolysis of myoglobin was used for different
experimental procedures. Examples of such tests
are the following: the test for protein
concentration determination using Bradford assay,
separation and identification of amino acid by thin-
layer chromatography, and qualitative color
reaction. Commented [3]: oi dagdagan mopa to

REFERENCES Commented [4]: pag mag aadd ka ng refernce,

ialphabetical order mona lang tapos paki italicize yung
mga title ng sources mo
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[2] Ammonium Sulfate Protein Precipitation: The

Key to Salting-Out
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[3] Isolation, Acid Hydrolysis & Color Reactions Of

Hydrolyzed Myoglobin
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solation-Acid-Hydrolysis-Color-Reactions-of-
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[4] Protein Precipitation Using Ammonium Sulfate

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[5] The Alkaline Hydrolysis Process

https://www.laboratoryequipment.com/article/20
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[6] THE HYDROLYSIS OF PROTEINS

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