1

ISOLATION, BASIC HYDROLYSIS, AND CHARACTERIZATION OF MYOGLOBIN

FROM MINCED BEEF SAMPLE
Mendoza, P., Mesina, K.*, Nodora, C., Nombre., M., Pineda, E.
Group 6, 2A Pharmacy
Department of Pharmacy, Faculty of Pharmacy
University of Santo Tomas

Abstract
Proteins are macromolecules composed of monomeric subunits termed as amino acids. The
transport protein myoglobin is usually observed in muscles and the reason for its red tinge. The
goals of the experiment are to execute the isolation and hydrolysis of proteins; perform
qualitative tests using color reactions and thin layer chromatography to determine the amino
acid components of the given protein sample; and make quantitative analysis using different
assay to acquire the protein concentration of the given myoglobin sample. The resulting solution
from the isolation of the myoglobin, a globular protein, showed a tinge of red as expected due
to the fact that the protein functions similarly to hemoglobin. The resulting solution from the
basic hydrolysis of the myoglobin sample produced a colorless solution with a slight precipitate.
All qualitative color reaction tests were done on both the intact protein and the hydrolyzed
protein to be able to compare results. Chromatography is a procedure that divides compounds
into their individual components and is done for the qualitative analysis of the amino acid
constituents from the hydrolyzed protein. The Bradford protein assay is based on the
proportional binding of the dye Coomassie to proteins and is used for the quantitative analysis
of proteins using the Beer-Lambert law, which states that there is a direct correlation between
absorbance and concentration. Some of the results acquired were accurate with the principles
involved in each experiment and their expected results, however, some were otherwise due to
the inevitable errors during the execution of the experiments.

Introduction transport proteins like myoglobin and
hemoglobin.
Proteins are macromolecules
composed of monomeric subunits termed
as amino acids. The human body is made up
of about 20% proteins and is found in every
individual cell. They are the most abundant
organic molecules present in living cells.
Protein in Greek means "of utmost
importance" and is often called as
workhorses of life for they provide the body
with structure and perform an immense
array of purposes such as proper immune
system function, digestion, hair and nail
growth, and more. Some classifications of Figure 2.1: The three dimensional structure
proteins are catalytic proteins or enzymes; of Myoglobin.
regulatory proteins such as the hormones
insulin and glucagon; structural proteins like The transport protein myoglobin is
collagen and elastin; defense proteins usually observed in muscles and the reason
known as the immunoglobulins; and for its red tinge. It carries and stores oxygen
for proper muscle function and can be
 2

separated from the muscle extract via the polarity of the compounds. This is also
ammonium sulfate precipitation. Myoglobin done for the qualitative analysis of the
is made up of almost solely -helices joined amino acid constituents from the basic
by short loops. It is a single-chain globular hydrolysate and is usually visualized using
protein of 153 amino acids connected by ninhydrin solution.
covalent peptide bonds. These bonds are
destroyed when the protein undergoes Several procedures can be
hydrolysis and result in a solution performed for the quantitative analysis of
containing amino acid fragments, which are proteins such as the Biuret test, Bradford
then called the hydrolysate. Acid, basic and assay, and Bicinchoninic acid (BCA) test.
enzymatic hydrolysis are the three most These are done to ascertain the protein
well-known types of hydrolysis. concentration from biological samples. The
Bradford assay is a colorimetric method for
Alkaline or basic hydrolysis is a determining protein concentration involving
process by which complex molecules are the binding of Coomassie dye to proteins in
split into their component by the acidic solution leading to an increased
introduction of ions of water (H2O), H+, and absorbance of the sample read at 595 nm
OH- within the atoms of the bonds. The using the spectrophotometers. The assay is
bases are usually water solutions of alkali sensitive to about 20 to 200 g protein. The
metal hydroxides like sodium hydroxide standard Bradford protein assay commonly
(NaOH) or potassium hydroxide (KOH). uses a range of 20-150 g protein; if only 1-
Heating the reactants dramatically hastens 10 g of protein per milliliter is used this
hydrolysis. would then be called the MicroBradford
protein assay. Quantitative analysis is
acquired using the Beer-Lambert law, which
states that there is a direct correlation
between absorbance and concentration.

Figure 2.2: The mechanism of alkaline/basic The objectives of this experiment
hydrolysis on the peptide bond between are to execute the isolation and hydrolysis
methionine (right) and alanine (left). of proteins; perform qualitative tests using
color reactions and thin layer
As stated, proteins are made up of chromatography to determine the amino
amino acids and certain functional groups acid components of the given protein
found in them can react to create sample; and make quantitative analysis
characteristically colored products. The using different assay to acquire the protein
qualitative color reactions include tests concentration of the given sample.
such as the Biuret test, the Ninhydrin test,
the Xanthoproteic test, Millons Test, the Methodology
Hopkins-Cole test, the Sakaguchi test, the
Nitroprusside test, Fohls test, the Test for A. Material(s) Used
Amides, and the Pauly test. These are done
For the Isolation of Myoglobin
to differentiate the different amino acid
1. Minced beef muscle
residues present in a protein sample.
2. 70% buffer-diluted (NH4)2SO4

solution, pH 7.5
Thin layer chromatography (TLC) is
3. Cheesecloth
a procedure done to separate and identify
4. Centrifuge tubes
compounds of interest and makes use of
 3

5. Centrifuge machine For the Separation and Identification of

6. (NH4)2SO4 crystals
Amino Acids by Thin-layer
Chromatography
For Alkaline hydrolysis of Intact Protein 1. 2% w/v of each amino acid
1. Isolated Myoglobin standard: tryptophan, arginine,
2. 4 M NaOH proline, cysteine, serine,
3. Hard glass test tube aspartice acid, tyrosine,
4. Autoclaving machine histidine, glycine, and alanine.
5. Distilled water 2. Acidic, basic, and enzymatic
6. 250-mL beaker hydrolysates
7. 1 M HCl 3. 1-Butanol:acetic acid:water
(4:1:5)
For Qualitative color reactions 4. 1% ninhydrin solution in spray
1. Intact protein solution bottle
(0.5g/0.5mL of protein in 1 mL 5. 12 x 5 cm TLC plate
distilled H2O) for each of the 10 6. Chromatography chamber (1-L
test tubes. beaker covered with a watch
2. 0.5 mL of the hydrolyzed glass)
sample for each of the another 7. Capillary tubes
separate 10 test tubes.
3. 2.5 M NaOH For the MicroBradford Protein Assay
4. 0.1 M CuSO4 1. BSA Standard Stock Solution
5. 0.1% Ninhydrin solution 2. Distilled water
6. Concentrated HNO3 3. Bradford reagent
7. Concentrated NaOH 4. 1.5-mL microcentrifuge
8. Millons reagent (Eppendorf) tubes
9. Hopkins-Cole reagent 5. 96-well microplate
10. Concentrated H2SO4 6. Micropipette
11. 10% NaOH 7. Spectrophotometer
12. 0.02% Naphthol solution
13. 2% NaOBr B. Procedure
14. 3 M NaOH
15. 2% Nitroprusside solution For the Isolation of Myoglobin
16. 30% NaOH In a small beaker, 0.6 g of the
17. 5% Pb(CH3COOH)2 minced beef and 6 mL of the 70% (NH4)2SO4
18. 20% NaOH solution was mixed together for a total of 1
19. 1% Sulfanilic acid minute for the myoglobin to be released.
20. 5% NaNO2 The dark-red extract was acquired by
21. 10% Na2CO3 expressing the mixture using the
22. Beaker cheesecloth and then transferred into the
23. Red and blue litmus paper centrifuge tubes. The extract was then
strips centrifuged at 13, 000 x g for 5 minutes. The
24. Hot plate supernatant liquid was transferred to
another empty centrifuge tube, adding and
mixing into it ~0.3-0.35 g of the fine
grounded (NH4)2SO4 crystals. The sample
was the centrifuged for 5 minutes and the
 4

resulting supernatant luquid was decanted

off. Millons Test
Five drops of the Millons reagent
For Alkaline hydrolysis of Intact Protein were added into a test tube with the
Ten milliliters of 4 M NaOH was intact protein solution and a separate
added to 0.5 g/0.5 mL of the isolated test tube with 0.5 mL of the hydrolyzed
protein in a hard glass test tube. The hard sample. The changes in color of the
glass test tube was the covered with cotton, two resulting solutions were recorded.
labeled, and autoclaved (for 5 hours at 15
psi). Ten milliliters of distilled water was Hopkins-Cole Test
added to the mixture after autoclaving and Twenty drops of the Hopkins-Cole
then transferred into a 250-mL beaker. One reagents were slowly added into a test
milliliter of 1 M HCl was added to neutralize tube with the intact protein solution
the mixture. and a separate test tube with 0.5 mL of
the hydrolyzed sample. Both test tubes
For Qualitative color reactions were inclined and 20 drops of the
concentrated H2SO4 were slowly added
Biuret Test along the sides of both test tubes and
Twenty drops of 2.5 M NaOH were were not shaken or mixed. The colors
added into a test tube with the intact of the interface of the two final
protein solution and a separate test solutions were recorded.
tube with 0.5 mL of the hydrolyzed
sample. Two to three drops of 0.1 M Sakaguchi Test
CuSO4 were added to each of the test Ten drops of the 10% NaOH and 10
tube. The colors of the two resulting drops of the 0.02% naphthol solution
solutions were recorded. were both added into a test tube with
the intact protein solution and a
Ninhydrin Test separate test tube with 0.5 mL of the
Six to ten drops of the 0.1% hydrolyzed sample. Both of the
ninhydrin solution were added into a mixtures were allowed to stand for a
test tube with the intact protein total of 3 minutes. Three drops of the
solution and a separate test tube with 2% NaOBr were then added into both
0.5 mL of the hydrolyzed sample. Both test tubes and was mixed. The colors of
test tubes were heated in a boiling the two final solutions were recorded.
water bath. The colors of the two
resulting solutions were recorded. Nitroprusside Test
Half milliliter (0.5 mL) of the 3 M
Xanthoproteic Test NaOH solution was added into a test
Ten drops of concentrated HNO3 tube with the intact protein solution
were slowly added into a test tube and a separate test tube with 0.5 mL of
with the intact protein solution and a the hydrolyzed sample. A quarter
separate test tube with 0.5 mL of the milliliter (0.25 mL) of the 2%
hydrolyzed sample. The colors of the nitroprusside solution was the added
two resulting solutions were recorded. to both of the test tubes. The colors of
Ten drops of concentrated NaOH were the two final solutions were recorded.
slowly added to each test tube. The
colors of the two final solutions were
recorded.
 5

Fohls Test inside the pre-equilibrated chamber and

Five drops of the 30% NaOH the solvent was set to be below the
solution and 2 drops of the 5% origin line. A watch glass was used to
Pb(CH3COOH)2 were added into a test cover the chamber and the solvent was
tube with the intact protein solution the allowed to ascend the plate
and a separate test tube with 0.5 mL of undisturbed. The plate was removed
the hydrolyzed sample. Both test tubes after the solvent front was
were place placed in a boiling water approximately 0.5 cm from the top edge.
bath. The appearances of dark brown The solvent front was marked with a line
or black sediments on both samples across. One percent ninhydrin reagent
were recorded. was the sprayed after allowing the plate
to dry. The plate was placed in the oven
Test for Amides for 1-3 minutes until the amino acid
One milliliter of the 20% NaOH was spots appear. The spots were encircled,
added into 10 mL of both samples. measured, and their Rf values
Both test tubes were then subjected computed.
into a boiling water bath. The presence
of the evolution of gas during heating For the MicroBradford Protein Assay
was tested on both using litmus paper Standard protein concentrations
mounted on the mouth of both test were prepared by mixing a certain
tubes. The change in colors of both amount of BSA Standard Stock Solution
litmus papers was recorded. and a corresponding amount of distilled
water and were then placed into six 1.5-
Paulys Test mL microcentrifuge (Eppendorf) tubes
The Diazo reagent was made by were labeled Blank, Standard 1,
adding together 3-5 drops of the 1% Standard 2, Standard 3, Standard 4, and
sulfanilic acid and 3 drops of the 5% Standard 5. Another 1.5-mL
NaNO2 solution. Five drops of both microcentrifuge tube was filled with the
samples and 3-5 drops of the 10% pure protein sample and labeled as
Na2CO3 were added into the diazo Unknown 1. A dilution of 1 L of the
reagent made. The appearance of a red hydrolysate sample in 99 L of distilled
coloration was recorded. H2O was filled into 1.5-mL
microcentrifuge tube and labeled
For the Separation and Identification of Unknown 2. Five microliters of the Blank,
Amino Acids by Thin-layer Standards 1 to Standard 5, Unknown 1,
Chromatography and Unknown 2 were transferred into a
The 12 x 15 TLC plate was prepared 96-well microplate using a micropipette.
and was marked with an origin line A dilution of the Bradford reagent was
across the plate having a 1.5-cm margin made by mixing 1 mL of the Bradford
from the bottom of the longer edge of reagent into 4 mL of distilled water and
the plate. Thirteen equidistant points 100 L of this dilution was then
were marked on the line for the spotting transferred into the wells containing the
of the amino acid standards and the Blank, Standards 1 to Standard 5,
hydrolysate sample. The standards were Unknown 1, and Unknown 2. The
applied 5 times and the samples 10 absorbance of the standards and
times using the capillary tubes allowing unknowns were read. Their
the spotting to dry after each concentrations against their absorbance
application. The plate is then placed were the graphed. The concentration of
 6

the unknown protein solution was named salting-in. At greater salt

determined using either graphical or concentrations, protein solubility usually
linear regression analysis. declines, leading to precipitation; this effect
is termed salting-out (Green & Hughes,
Results and Discussion 1955). A salt's capability to cause selective
precipitation is dependent on various
The following tables below show interactions with the water and solutes. As
the observations acquired from the results (NH4)2SO4 has much a higher solubility than
of the isolation, hydrolysis, and color any of the phosphate salts, it is the reagent
reactions of the protein sample followed by of choice for salting-out.
the tables showing the measurements or
values that were computed from the thin- Table 2.2: Alkaline/Basic Hydrolysis of
layer chromatography and total protein Intact Protein data.
assay as well as the graph for the standard Mode of Description of the
curve of the myoglobin sample. Hydrolysis Hydrolysate
Basic Colorless solution with slight
Table 2.1: Isolation of Proteins data. precipitate
Protein Description
Myoglobin Blush-red colored solution The resulting solution from the
basic hydrolysis of the myoglobin sample
The resulting solution from the produced a colorless solution with a slight
isolation of the myoglobin, a globular precipitate. Hydrolysis is a reaction
protein, showed a tinge of red as expected comprising the rupture of a bond in a
due to the fact that the protein functions molecule using water. The reaction chiefly
similarly to hemoglobin. Both act as a happens between an ion and water
carrier and distribute oxygen to the molecules and usually alters the pH of a
different parts of the body. Myoglobins solution.
molecular constituent termed heme
contains iron giving it its signature red-
brown color.

Figure 2.4: Hydrolysate from the basic
hydrolysis of the myoglobin sample.


Figure 2.3: Isolated myoglobin from the One of the advantages of using
minced beef muscle. basic hydrolysis is that tryptophan is not
destroyed during the process; however,
The solubility of globular proteins arginine, asparagine, glutamine, and serine
rises upon the addition of salt, an effect are damaged.

 7

Table 2.3: Qualitative Color Reactions data.

Color Intact Hydrolyzed
Reaction Protein Protein
Basic
Biuret ( + ) ( + )
Ninhydrin ( + ) ( - )
Xanthoproteic ( - ) ( - )
Millons ( + ) ( - )
Hopkins-Cole ( - ) ( - )
Sakaguchi ( + ) ( - )
Nitroprusside ( + ) ( - )
Fohls ( + ) ( - ) Figure 2.5: Color reaction results of the
Test for ( + ) ( + ) intact protein. The results from left to right:
Amides R to B | B to B R to B | B to B from Biuret, Ninhydrin, Xanthoproteic,
Millons, Hopkins-Cole, Sakaguchi,
Pauly ( - ) ( - )
Nitroprusside, Fohls, Test for Amides, and

Paulys.
Various color-producing reagents

(dyes) are used to qualitatively detect the

pres-ence of certain functional groups of
amino acids found in proteins. The
qualitative color reactions include tests
such as the Biuret test, the Ninhydrin test,
the Xanthoproteic test, Millons Test, the
Hopkins-Cole test, the Sakaguchi test, the
Nitroprusside test, Fohls test, the Test for
Amides, and the Pauly test. These tests are
for the determination of: the presence of
peptide bonds, an alpha amino acid,
aromatic amino acids (Trp, Phe, and Tyr),
tyrosine residues, tryptophan residues,
Figure 2.6: Color reaction results of the
guanidines (Arg), thiol group (Cys), sulfur
hydrolyzed protein. The results from left to
containing (Cys and Met), amides (Arg and
right: from Biuret, Ninhydrin,
Gln), and histidine and tyrosine
Xanthoproteic, Millons, Hopkins-Cole,
respectively. Positive results on these tests
Sakaguchi, Nitroprusside, Fohls, Test for
would show: blue to blue-violet solution,
Amides, and Paulys.
blue to blue-violet solution or yello solution

for proline, yellow in the addition of the
All qualitative color reaction tests
acid whilst orange in the addition of the
were done on both the intact protein and
base, red/rose/salmon color, purple
the hydrolyzed protein to be able to
coloration of the interface, red/wine color,
compare results. From the result of the
red color, black or brown precipitate,
reactions on the intact protein all tests
evolution of gas indicated by the change in
showed a positive visible result except for
color of the litmus paper strip from red to
the Xanthoproteic, Hopkins-Cole, and
blue or blue to blue, and red solution
Paulys tests which showed otherwise. From
respectively.
this we can infer that the intact protein

sample used lacked aromatic amino acids
(Trp, Phe, and Tyr), tryptophan residues,
 8

and histidine and tyrosine residues. of 1-Butanol:acetic acid:water in 4:1:5 ratio

Comparing it to the result acquired from would be polar. The polar compounds will
the reactions on the hydrolyzed protein be more strongly attracted to the mobile
with all tests being negative with the phase or the solvent and will move faster
exception of the Biuret test and Test for and will appear closer to the top of the
Amides. From this we could infer that there plate. On the other hand, Non-polar
are still peptide bonds present in the compounds will be more attracted to the
hydrolyzed sample, which should not be the TLC plate and will spend less time in the
case because hydrolysis disrupts peptide moving phase thus appearing lower on the
bonds. We can also observe that the Test plate.
for Amides showed a positive result hinting
the presence of arginine and glutamine
contrary to the principle that during the
process of hydrolysis arginine, asparagine,
glutamine, and serine are damaged.

Table 2.4: Separation and identification of
Amino Acids by Thin-layer Chromatography
data.
Rf Values of
Amino Acid Standard the Spot
Base Figure 2.7: The TLC plate results.
Hydrolysate
Several compounds move in
Tryptophan 0.51
different distances and with different speed
Arginine 0.36
on the plate by capillary action. To take this
Proline 0.37
variation into account, the ratio of the two
Cysteine 0.37
distances (distance by the compound and
Serine 0.37 0.47
distance by the solvent front) is calculated
Aspartic Acid 0.34 and reported. This ratio is called the
Tyrosine 0.32 retention factor (Rf). Due to lack of time for
Histidine 0.32 the experiment, the solvent was not
Glycine 0.31 allowed to rise up to the expected
Alanine 0.29 measurement at the top of the TLC plate,
thus giving unreliable values for the
Chromatography is a procedure retention factors of the compounds.
that divides compounds into their individual
components. The principle associated with Table 2.5: MicroBradford Total Protein
the separation of the amino acid Assay data.
components on the paper is polarity. In the Test Tube/ Protein
thin-layer chromatography, the stationary Standard Concentration Absorbance
phase is a highly uniform absorbent paper. (mg/mL)
Cellulose (nonpolar and a polymer of the Blank 0 0.106
simple sugar, glucose) in the form of paper Std. 1 0.01 0.104
sheets creates a fitting medium where Std. 2 0.02 0.102
various components are absorbed by the Std. 3 0.03 0.106
fibers thus the components will visibly
Std. 4 0.04 0.112
separate. The mobile phase being a mixture
Std. 5 0.05 0.116
 9

Unknown 1 0.1 0.118 These experiments were conducted

Unknown 2 0.001 0.132 as an application of the knowledge on the
principles involved in each of tests and
The Bradford protein assay is one of procedures done. Some of the results
the numerous simple methods usually used gathered were coherent and accurate with
to define the total protein concentration of the said principles and their expected
a sample. The process is based on the results although some were otherwise due
proportional binding of the dye Coomassie to the inevitable errors during the
to proteins. The dyes attach to proteins by execution of the experiments or the lack of
ionic interactions between dye sulfonic acid time given.
groups and positive protein amine groups
as well as through Van der Waals References
attractions. The standard Bradford protein
assay commonly uses a range of 20-150 g Bathan, G., Bayquen, A., Cruz, C.,
protein; if only 1-10 g of protein per Crisostomo, A., De Guia, R., Farrow,
milliliter is used this would then be called F., Pea, G., Torres, P. (2014).
the MicroBradford protein assay. Laboratory manual in Organic
Chemistry (Revised ed.). Quezon
City, Philippines: C & E Publishing,
Inc.

Bathan, G., Crisostomo, A., Daya, M., De

Guia, R., Farrow, F., Gabona, M.,
Guevarra, L., Ysrael, M. (2017).
Laboratory manual in General
Biochemistry (2nd ed.). Quezon City,
Philippines: C & E Publishing, Inc.

Campbell, M., & Farrell, S. (2013).

Biochemistry (8th ed.). Connecticut,

Figure 2.8: Protein concentration vs. USA: Cengage Learning
Absorbance data.
Green, A., & Hughes, W. (1995). Protein

solubility on the basis of solubility in
The Bradford assay is linear over a
aqueous solutions of salts and
low range, from 0 g/mL to 2000 g/mL,
organic solvents. Methods Enzymol
therefore making dilutions of a sample
(1), 6790
necessary before an examination. In making
these dilutions, error in one dilution
Kaye, G., Weber, P., & Wetzel, W. (2004).
coalesces in extra dilutions ending in a
The Alkaline Hydrolysis Process.
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 10

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Three Dimensional Structure of Myoglobin

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