UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY
MANILA, PHILIPPINES

ISOLATION AND CHARACTERIZATION OF PROTEINS

Manalo, P.R., Quibol, S. M., Raagas, E., Rosario, C.F., Tabula, M.C., Tan, J.M., Tornea, E.M.

ABSTRACT
Proteins are large molecules composed of amino acids obtained from various sources. The main
objective of the experiment is to determine the presence of proteins in milk, wheat flour, and beef
muscle, and to isolate the following proteins: casein, albumin, gluten, and myoglobin. Different isolation
methods were utilized throughout the experiment for the different kinds of proteins. This involved the
hydrolysis principle where the intact proteins obtained from the initial isolation were used and treated
as acidic, basic, and enzymatic hydrolysates. Qualitative color reactions were noted to determine the
positive and negative results. Amino acids were separated and identified through their differences
based on their polarities. For this identification, the thin-layer chromatography was used with the aid
of the BAW solution or 1-Butanol: acetic acid: water ratio. Several qualitative color reactions exhibited
a positive result for intact casein and casein enzymatic hydrolysate. However, results from the color
reactions cannot be confirmed with the result of TLC of the casein enzymatic hydrolysate which
provided fragmentations of peptide chains only. Despite the limited amino standards used, results of
color reactions of gluten signified that it may contain tyrosine, phenylalanine, histidine, cysteine,
arginine and methionine. In accordance to this, the TLC results confirmed that gluten contained some
of these amino acids where non-polar substances were found to exhibit higher Rf values with higher
affinity with the solvent given. For the quantitative protein analysis of albumin, the concentration
exhibited direct relationship with absorbance. However, protein quantitation may exhibit selective
determination to specific amino acids.

INTRODUCTION of Albumin from Skimmed Milk with the use of

Protein is a large molecule composed of
different amino acids and may be obtained from
various sources. Protein isolation involves
processes that permits isolation of proteins from
a complex mixture. Protein isolation and
purification depends on the protein's
physicochemical characteristics such as
structure, molecular weight, solubility,
isoelectric pH, and heat stability. On the other
hand, they are also separated according to size,
shape, charge, and hydrophobicity. The most
commonly used methods are heat denaturation,
solubilization, isoelectric precipitation, and
chromatography.
Different isolation methods were utilized
throughout the experiment. First is the isolation
of Casein from Skimmed Milk, wherein an acid
was used to adjust the pH to 4.6, obtaining the
isoelectric pH of casein. Second is the isolation
denaturation and coagulation of heat. Third is
the isolation of Gluten from Wheat Flour through
the removal of starch (washing the dough with
water; results to the insoluble gluten when
starch is removed). Fourth is the isolation of
Myoglobin from Muscle using ammonium sulfate
precipitation on the buffered muscle extract.
Intact Proteins were also isolated through
Hydrolysis, to obtain information about the
composition of the sample. This process can be
carried out by treating the protein with alkali,
acid, or proteolytic enzymes.
For the acid hydrolysis, with the presence of 6M
HCl, the secondary, tertiary and quaternary
structures of the casein are lost. This allows
covalent bonds of the amino acids to break,
leading to a complete hydrolyzation of the
peptide bonds. The characterization of peptide
chains was determined through the use of
qualitative color reaction tests which includes
Biuret test (detects the presence of peptide
bonds), Ninhydrin test (determination of alpha
amino acids), Xanthoproteic test (determines
the side chain of aromatic amino acids), Millon’s
and Hopkins-Cole (determines tyrosine and
tryptophan residues), Nitroprusside and Fohl’s
test (determines presence of sulfur-containing
amino acids), Sakaguchi test (for arginine),
Pauly test (for histidine and tyrosine) and lastly,
the Amide test (detects R-groups of glutamine
and asparagine.
Paper chromatography was involved as a
method for the separation and identification of
amino acids based on their polarities.
Components of a sample mixture separate into
the mobile phase and stationary phase as a
result of their differential affinity where their
polarity can be further determined.
Different quantitative tests for Protein Analysis,
including the Biuret test, Bradford assay, Lowry
assay, and Bicinchoninic acid, were used to
react with samples to produce colors to be
observed using a Spectrophotometer. Results
were analyzed using the Beer-Lambert Law.
In this experiment, the objectives are to perform
qualitative tests on amino acids contained in
intact and hydrolyzed proteins and perform acid,
alkaline, and enzymatic hydrolysis on the
isolated proteins to be obtained. Through these
processes, the amino acid components of
proteins and their quantitative concentration will
be determined using the thin-layer
chromatography.
METHODOLOGY
Different samples were used for the experiment,
including tryptophan, arginine, proline, cysteine,
serine, aspartate, histidine, glycine, alanine,
Casein, acid hydrolysate. These samples were
then utilized in different tests for the isolation of
proteins: hydrolysis, qualitative and quantitative
protein analyses, and separation and
identification of amino acids by thin-layer
chromatography.
Isolation of Casein from Skimmed milk
Powdered, non-fat milk was obtained and 20
grams of which was placed in a 100 mL beaker.
It was then added with 50 mL of distilled water
with continuous mixing. The mixture was heated
using a hot plate with a temperature of 40°C. A
thermometer was placed in the mixture to
monitor and assure that the temperature will not
exceed 40°C. The addition of 10% acetic acid
(dropwise) was done as the mixture reached
40°C. Gradual stirring was done after every 5
drops with continuous addition of acetic acid
until curding was observed.
The mixture was then filtered, and the resulting
residue was dried and weighed. The remaining
filtrate was transferred to a test tube and was
set aside for the albumin isolation and analysis.
Isolation of Albumin from Skimmed Milk
The filtrate that was obtained from the isolation
of casein from skimmed milk was used. Half of
the filtrate was transferred to a beaker while the
remaining half was set aside for quantitative
protein analysis. Using a hot plate, the heating
of filtrate, contained in the beaker, was done for
5 minutes at 75°C. Decantation of the liquid was
done from the precipitated albumin.
Isolation of Gluten from Wheat Flour
A cup of wheat flour was obtained and mixed
with enough distilled water to make a thick
dough. The dough was then wrapped with a
cheesecloth and washed with a tap water. Every
washing was collected and tested for presence
of starch using iodine solution. Washing of
dough was done repeatedly until a negative
result for starch was obtained. The insoluble
material was set aside for hydrolysis and
qualitative protein analysis.
Acid hydrolysis of Gluten
The isolated Gluten was weighed, and 0.5 grams
was transferred to a hard glass test tube. It was
added with 5 mL of 6M HCl and covered with
cotton. The sample was autoclaved for 5 hours
(15 psi).
After autoclaving, 2 mL aliquot of the hydrolyzed
gluten was transferred to a test tube with the
addition of 10 mL distilled water. The sample
was neutralized with 1M NaOH.
Alkaline hydrolysis of Gluten
The isolated Gluten was weighed, and 0.5 grams
of it was transferred to a hard glass test tube,
with the addition of 10 mL of 6M NaOH. The
hard glass test tube was covered with cotton
and was autoclaved for 5 hours (15 psi).
After autoclaving, 2 mL aliquot of the hydrolyzed
gluten was transferred to a test tube and was
added with 10 mL distilled water. The sample
was neutralized with 1M HCl.
Enzymatic hydrolysis of Casein
The isolated casein was placed in a beaker.
Then, 10 mL of 1-2% protein solution in buffer
and 5 mL saturated protease solution were
added. The mixture was incubated using a
water bath at 35°C-40°C for 60 minutes.
Qualitative Color Reactions
The intact casein, intact gluten, gluten acid
hydrolysate, gluten alkaline hydrolysate, and
enzymatic casein hydrolysate were the samples
used for the following tests.
The weighed intact protein sample (0.5 grams)
was added with 1 mL distilled water and 1 mL
of hydrolyzed sample. These samples were
prepared and placed in a microcentrifuge tube.
A. Sakaguchi Test
The sample was added with 50 mcL of 10%
NaOH and 50 mcL of 0.02% of napthol solution.
The solution was mixed and stood for 3 minutes.
It was then added with 20 mcL of 2 % NaOBr.
The solution was mixed, and the color of the
solution was noted.
B. Biuret Test
The sample was treated with 400 mcL of 2.5M
NaOH and was mixed before the addition of 20
mcl of 0.1M of CuSO4. Constant shaking was
done until a change in color was visible. Color
change was noted.
C. Ninhydrin Test
For this test, 50 mcL of 0.1% of ninhydrin
solution was mixed to the sample. Then,
shaking and heating of the mixture was done in
a boiling water bath. The color of the reaction
was noted.
D. Hopkins-Cole Test
The Hopkin-Cole reagent (100 mcL) was mixed
to the sample and placed in a microcentrifuge
tube. In an inclined position, slow addition of 20
drops of concentrated H2SO4 was done,
touching only the side of the tube. The interface
was observed and recorded.
E. Nitroprusside Test
For his test, the reagents used were 3M NaOH
and 2% nitroprusside solution. The sample was
first added 250 mcL of 3M NaOH, followed by
the addition of 250 mcL of 2% nitroprusside
solution. The resulting color of the solution was
recorded.
F. Millon’s Test
Into 1 ml of the sample, 50 mcL of millon’s
reagent was added. The resulting color of the
solution was observed and recorded.
G. Test for Amides
The sample was added with 50 mcL of 20%
NaOH. In a boiling water bath, the mixture was
heated and tested for the evolution of gas using
a litmus paper that was placed over the mouth
of the microcentrifuge tube. Results were
observed and recorded.
H. Xanthoproteic Test
The sample was treated with 10 drops of
concentrated HNO3 and was mixed well. A
change in color was immediately observed. After
which, slow addition of 10 drops of concentrated
NaOH was done. The solution was mixed and
was observed further for color change.
Quantitative Protein Analysis
The protein concentration of the samples was
determined using different techniques including
the Biuret test, Bradford and MicroBradford
assay. The quantitative analysis of the biological
samples was expressed using the Beer-Lambert spectrophotometer, the absorbance of the
Law. samples was determined and was graphed with
the use of the graphical or linear regression
A. Biuret Total Protein Assay analysis.
Biological samples and protein standards were
prepared by mixing solutions according to what Thin-Layer Chromatography
is shown in Table 1. The unknown protein There were 10 amino acids used as standards in
solution samples were diluted with distilled 2% w/v for this test: Tryptophan, Arginine,
water to produce samples with the ratios of Proline, Cysteine, Serine, Aspartic acid,
1:100 and 1:000. Tyrosine, Histidine, Glycine and Alanine. Three
samples were used for separation and
Table 1. Standard Protein Concentrations identification of its amino acids: enzymatic
for Biuret Assay casein hydrolysate, gluten acid hydrolysate and
BSA gluten alkaline hydrolysate.
Standard
Test Tube Distilled The TLC plate was prepared, with its origin
Stock
(mL) Water drawn using a pencil. The origin was drawn
Solution
(mL) across the plate, 1.5 cm away from the longer
Blank 0 2.50 edge of the plate. Thirteen equidistant points
Standard 1 0.10 2.40 were then marked at the origin. Using a capillary
Standard 2 0.50 2.00 tube, each sample was applied and drying of the
Standard 3 1.00 1.50 samples was done every after application. Used
Standard 4 1.50 1.0 capillary tube was replaced after every sample.
Standard 5 2.50 0
The solvent used was 1-Butanol: Acetic acid:
As described in Table 1, the blank and standard water (4:1:5). Inside the chamber, enough
protein solutions was prepared through solvent was placed in a level below the origin of
transferring 2.50 mL of the 1:100 diluted the TLC plate, carefully placed inside. The
unknown protein into another test tube. A 2.5 chamber was covered, and the solvent was
mL Biuret reagent was added to each test tube allowed to rise undisturbed. The removal of the
and were heated in a 40°C-50°C water bath for plate from the chamber was done when the
5 minutes. Before transferring to the cuvettes, solvent front has traveled approximately 0.5 cm
the heated solutions were first cooled and made from the top edge. The distance traveled by the
ready for the placement in the solvent front was marked using a pencil followed
spectrophotometer. For this test, the by the air-drying of the chromatogram through
wavelength was set to 549 nm with the reading spraying of 1% ninhydrin solution. The
set to 0 using the blank. The absorbance chromatogram was placed in an oven for 1-3
reading of the standard and samples were minutes and visible spots were identified and
noted. Using the graphical or linear method, the measured for its Rf values.
concentration of the unknown protein solution
was determined. RESULTS AND DISCUSSION
Different methods and tests proceeding the
B. Bradford Total Protein Assay isolation of proteins were utilized with the
samples. Hydrolysis, qualitative and quantitative
The standard protein solutions were prepared as
protein analyses, and separation and
noted in Table 1. A total of 2.50 mL of the
identification of amino acids by thin-layer
diluted unknown protein solution was
chromatography were done to satisfy the need
transferred into another test tube. For each test
to accomplish the objectives of the experiment.
tube, 2.5 mL of Bradford reagent was added and
was left standing for 5 minutes. Using the
Isolation of Proteins
Casein and albumin are globular proteins found
in milk. Casein was isolated by the addition of
acetic acid on the mixture at 40°C. It
precipitated as a white, curdy solid particle
which implied that the mixture reached a pH of
4.6, the isoelectric pH of casein, wherein the
protein becomes insoluble. The filtrate of this
mixture was heated to 75°C for 5 minutes and
a very small amount of albumin precipitated,
also appearing as a white solid particle. Gluten
from wheat flour was isolated by washing the
dough with water. An insoluble material, gluten,
was collected which appeared as a tough and
elastic solid substance.
Hydrolysis of Proteins
To obtain information about the composition of
the protein, hydrolysis was carried out by
treating the protein with acid, alkali or
proteolytic enzymes.
To produce the acid hydrolysate, 5ml of 6M HCl
was added to 0.5 g of gluten. On the other hand,
an alkaline hydrolysate was produces by adding
10 mL of the 4M NaOH to 0.5 g of gluten.
Proceeding to autoclaving, the acid hydrolysate
produced a dark brown solution with particles
while the alkaline hydrolysate exhibited a turbid
or cloudy white solution. Both the acid and
alkaline mixture were neutralized using 1M
NaOH (for the acid mixture) and 1M HCl (for the
alkaline mixture) for the characterization tests
and chromatography. The enzymatic
hydrolysate, after autoclaving, appeared clear
and was found to have an aroma of cheese.
Qualitative Color Reactions
Different tests were performed to characterize
free amino acids and proteins by reacting to
chemically reactive groups of amino acids. The
results of the qualitative test for each sample
are shown in Table 2.

Table 2. Qualitative Color Reactions of Samples

Visible Results
Intact Gluten acid Casein enzymatic
Color Test Intact casein
gluten hydrolysate hydrolysate
Biuret Test Violet Light violet Bluish gray Light violet
Ninhydrin Test No color change Colorless No change Clear solution
Xanthoproteic Very light
Yellow Yellowish color Yellow
Test yellow
Cloudy with white
Millon’s Test No change Colorless No change
ppt
Hopkins-Cole Formation of Brown ring with
Colorless No change
Test purple ring layer
Sakaguchi Test No color change Light pink Light orange Pink
Nitroprusside
No color change Bright yellow Bright yellow Yellow
Test
Appearance of Appearance of dark
Fohl’s Test Colorless Light brown
dark sediments sediments
Evolution of
Test for Amides Evolution of gas No change Evolution of gas
gas
Pauly’s Test Red orange Red orange Red Red
Biuret Total Protein Assay Separation and Identification of Amino
Biuret test is used to detect the presence of Acids by Thin-Layer Chromatography
protein in a sample. All samples gave a positive The amino acids contained in different protein
result in this test, giving a violet coloration of hydrolysates were identified through thin-layer
the mixture. chromatography. After being placed inside the
oven for 3 minutes, amino acid constituents
Ninhydrin test was used to confirm the presence appeared as spots, shown in the figures below.
of a free -NH2 group in an amino acid and only
the alkaline hydrolysate of myoglobin produced
a positive result (blue violet) for this test. All
samples showed positive results for
xanthoproteic test with yellow coloration, which
implies that they contain amino acids carrying
an aromatic group. For Millon’s test, a red
solution or precipitate indicates a positive result
which means that a phenol group is present in
the amino acids. All samples produced a
negative result for this test. As for the Hopkins- Figure 1. TLC Chromatograph of 10
Cole test, it was used to test for the presence of Amino Acids
tryptophan in proteins indicated by a deep blue
or purple coloration. For this test, no sample
was confirmed of having this amino acid. For
Sakaguchi test, none of the samples produced a
red-colored complex (positive), indicating the
presence of arginine. Thus, the samples
produced negative results for this test. The
nitroprusside test was used to detect the
presence of cysteine or thiol groups in proteins,
giving a purple hue coloration and thus,
indicating a positive result. For this test, no Figure 2. TLC Chromatograph of Isolated
sample was confirmed to contain cysteine Proteins
(negative). Fohl’s test, on the other hand was
used to detect the presence of sulfur containing The Rf values of amino acids and isolated
amino acid and only the acid hydrolysate of proteins were calculated by dividing the distance
gluten gave a positive result by producing a moved by the spot by the distance moved by
brown coloration. All samples, except for the the solvent front. The calculated Rf values for
acid hydrolysate of gluten, yielded a positive each amino acid and isolated protein are shown
result for the test for amides by showing an in Table 3 and Table 4.
evolution of gas confirmed through the change
of litmus paper from red to blue. Lastly, Pauly’s In thin layer chromatography, the separation of
amino acids is determined by their polarities and
test was utilized to detect the presence of
the affinity of each to the mobile and the
tyrosine or histidine in proteins. All samples
stationary phases. The mobile phase is
gave a positive result for this test, producing a described as the solvent used to analyze the
red coloration of the mixture. substance or the 1-Butanol: Acetic acid: water
(4:1:5) while the stationary phase is the
medium used where the separation occurs or
the TLC plate. Based on Table 3, the Rf values
for the amino acids concludes that Tryptophan
travelled the farthest from the base line. This is
because it is a non-polar or hydrophobic amino
acid and is highly soluble in the eluting solvent.
The more soluble it is, the higher affinity it has
for the mobile phase which denotes that if an
amino acid in turn has a higher affinity for the
stationary phase, it moves slower than the
solvent front. With this, Histidine, a polar amino
acid, has higher affinity to the stationary phase
which made it travel the least from the base line.
Butanol is a non-polar solvent that aided the
non-polar amino acids up the chromatogram
while acetic acid aided the polar amino acids.
The non-polar amino acids traveled farthest
from the base line because it is soluble in the
eluting solvent while the polar amino acids
travelled the least. However, it should be noted
that solubility is very dependent on the pH of
the solvent. Since the solvent mixture contains
4 parts of butanol for every 1 part of acetic acid,
the results favored the non-polar amino acids.
Ninhydrin solution was sprayed to the TLC plate,
to identify the amino acids by producing a blue
violet color.

Table 3. Standard Rf values of Amino Acids

distance moved by spot
Amino Acid Rf value
distance moved by solvent front
Alanine (A) 14/54 0.26
Arginine (R) 8/54 0.15
Aspartic Acid (D) 9/54 0.17
Cysteine (C) 13/54 0.24
Glycine (G) 11/54 0.20
Histidine (H) 6/54 0.11
Proline (P) 11/54 0.20
Serine (S) 10/54 0.19
Tryptophan (W) 30/54 0.56
Tyrosine (Y) 24/54 0.44

Table 4. Rf values of Isolated Proteins

distance moved by spot
Isolated Protein Rf value
distance moved by solvent front
Gluten acid
25 cm/50 cm 0.50 cm
hydrolysate
Gluten alkaline
23 cm/50 cm 0.46 cm
hydrolysate
Casein enzymatic
- 0 cm
hydrolysate
Comparing the resulting rf values, it can be may exhibit selective determination to specific
inferred that gluten acid hydrolysate is most amino acids.
likely to contain tryptophan, tyrosine, alanine,
histidine or arginine. For gluten alkaline REFERENCES
hydrolysate tyrosine and arginine are most likely • https://www.macalester.edu/~kuwata/Clas
the amino acids that are contained in the ses/2001-
hydrolysate. No dark spots appeared for the 02/Chem%2011/Revised%20Amino%20Aci
casein enzymatic hydroxylate, thus, no amino ds%20(9%201%2001).pdf
acid can be confirmed of being a component of • Isolation and Characterization of Proteins
the isolated protein. Formal Report. (n.d.). Retrieved from
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CONCLUSION 185/Isolation-and-Characterization-of-
Casein is a protein composed of the amino Proteins-Formal-Report.
acids: glutamic acid, proline and isoleucine,
lysine, leucine, valine, aspartic acid, serine,
tyrosine, phenylalanine, threonine, arginine,
histidine, alanine, methionine, glycine,
tryptophan and cysteine. Several qualitative
color reactions exhibited a positive result for
intact casein and casein enzymatic hydrolysate.
However, results from the color reactions
cannot be confirmed with the result of TLC of
the casein enzymatic hydrolysate as it provided
fragmentations of peptide chains and not free
amino acids. Thus, it did not exhibit any spot for
determination and identification.

Gluten is an insoluble protein from wheat that

contains several amino acids. The results of
color reactions of gluten signify that gluten may
contain the amino acids: tyrosine,
phenylalanine, histidine, cysteine, arginine and
methionine. The TLC results confirm that gluten
contains some of the aforementioned amino
acids. Comparison of the results were limited as
only 10 amino acid standards were used in TLC
preparation.

Thin layer chromatography was used to

separate and identify amino acids. This method
was based on the polarities of the samples
where the non-polar substances exhibited
higher Rf values possessing higher affinity with
the solvent given.

Furthermore, results of the quantitative protein

analysis of albumin can conclude that
concentration exhibits direct relationship with
the absorbance. However, protein quantitation