Isolation and Characterization of Proteins

Ayeessa Marie Janiella U. Alvar, John Marlon P. Ancheta, Jannzel Marie

Bianca T. Ang, Jaira Flor B. Bartilad and Kyle Bernadette A. Belleca
Group 1 2C Medical Technology Biochemistry Laboratory

ABSTRACT
The intact protein, gluten, is isolated from wheat flour by difference in solubility using a cheesecloth
and running water. Gluten is a family of proteins found in grains like wheat, rye, spelt and barley.
Isolated gluten had a dirty yellow color with gum-like consistency. It was subjected to characterization
by qualitative reaction tests and by paper chromatography. The group was assigned to perform acid
hydrolysation on .5g of the intact protein, the result of which is a hydrolyzed protein. A hydrolyzed
protein is a protein that has been broken down into its component amino acids. The hydrolysate of
gluten was a brown solution. The intact protein and the hydrolyzed protein were both used as protein
samples for the reaction tests and for paper chromatography. The qualitative reaction tests are used
for characterizing both free amino acids and proteins. The qualitative reactions tests that were
performed were the following: Biuret test, Ninhydrin test, Xanthoproteic test, Millon’s test, Hopkins-
Cole test, Sakaguchi test, Nitroprusside test, Fohl’s test, Amide test, and the Pauly’s test. These tests
are for general tests for alpha amino acids, and are for examining the presence of specific amino acid
side chains.
INTRODUCTION different methods are used, but the most
Proteins are found throughout the common are isoelectric precipitation, heat
body, it can either found in the muscle, denaturation, solubilization, salt-induced
bone, hair, skin or any body part [2]. It is precipitation, chromatography, and ultra-
a class of compound which serves as an centrifugation [3].
important component of the cell. They are In this experiment, the group was
polymers of twenty naturally occurring tasked to isolate gluten from wheat flour.
amino acids. The sequence and Gluten is a mixture of two proteins,
organization of every amino acid is the glutenin and gliadin. It is also the
reason for the unique physico-chemical composite of a prolamin and glutelin,
characteristics of a protein. In order to which exist with starch in the endosperm
determine the components of a protein, of various grass-related grains. Gluten is
isolation and purification techniques are described to be yellowish-white, tough,
used [1]. Protein isolation is a series of elastic, and sticky protein. It is also said
process designed to isolate one or few to be a long molecule having strong and
proteins from a complex mixture. The flexible characteristics, this is the reason
isolation process aims to characterize the why gluten became a useful ingredient in
molecular structure, molecular weight, bread making. Gluten has the capability to
solubility in different solvents, isoelectric trap the carbon dioxide from the reaction
pH, heat stability, and hydrophobicity of a of flour and yeast. It also gives flour the
specific protein [1,7]. The isolation and chewy characteristic making it rise and
characterization of proteins subjects the helps the dough keep its shape. In order
biomolecules to undergo denaturation. for the gluten to be isolated, the
Denaturation of proteins involves the difference in solubility is taken into
disruption and possible destruction of the consideration. The insoluble protein is
secondary, tertiary and quaternary obtained by removing the soluble protein
structure, but is not strong enough to through washing.
break the primary structure [3]. It Hydrolysis is a chemical process of
functions by a chemical interactant or decomposition involving the splitting of a
factor, and they are change in heat, bond and the addition of the hydrogen
change in pH, subjection to detergent, cation and the hydroxide anion of water
and processes which can fragment [3]. It aims to break the peptide bonds
disulfide bonds [3]. In the laboratory, which will result to smaller amino acid
chains, and free amino acids. The solution for autoclaving. After autoclaving the
containing the protein is called a mixture, the group record its appearance.
hydrolysate. Once the hydrolysate is 10 mL of distilled water was added and
prepared, different color reactions are the mixture was transfer into a 250-mL
performed. The color reactions help beaker. Lastly, the mixture was
determine the presence of the different neutralized with 1 M NaOH then was used
amino acids in gluten. as the sample for the characterization
tests and chromatography.
EXPERIMENTAL
A.Test Compounds Used 3. Alkaline Hydrolysis of intact protein
The sample compounds used in The procedure for alkaline
this experiment were the intact and hydrolysis is almost the same for alkaline
hydrolyzed protein samples, 6M NaOH, 3M hydrolysis. The main difference is that
NaOH, 2.5M NaOH, 20% NaOH, 10% instead of adding 5 mL of 0.6 M HCl into
NaOH, Conc. NaOH solution, 5% NaNO2, the 0.5 g of the isolated protein, 10 mL of
10% Na2NO3, 2% NaOBr, 0.1M CuSO4, 4 M NaOH was added. It was also labeled
5% (CH3COO)2Pb, 2% nitroprusside the same way however, H+ hydrolysis
solution, .002% napthol solution, 1% was replaced with OH- hydrolysis. Besides
sulfanilic acid, 0.1% ninhydrin solution, these two differences, the procedure is
Conc. HNO3, Conc. H2SO4, Hopkins-Cole the same with acid hydrolysis.
reagent, Millon’s reagent, red and blue
litmus paper strips, Beaker, Hot plate 4. Enzymatic hydrolysis of intact protein
For enzymatic hydrolysis, 100 mL of
B. Procedure distilled water protein mixture was prepared. 10
1. Isolation of gluten from wheat flour mL of the protein mixture was taken and mixed
with 10 mL of saturated protease solution.
The isolation of gluten from the
Alternatively, 0.50 g of protease may also be
wheat flour was done by adding an ample
mixed directly with 50 mL of the protein mixture.
amount of water to 250 grams of wheat Subsequently, 10 mL of the 0.1 M phosphate
flour. After kneading it to the right buffer with pH 7.5 was added. The mixture was
consistency, it was wrapped with a placed in a water bath for 60 minutes with a
cheesecloth and washed under running temperature ranging from 35℃ to 40℃
water. The continuous washing of the depending on the source of enzyme. The
dough removes all the starch and the mixture was cooled before using in the
insoluble material found inside the procedure for qualitative color reactions and for
cheesecloth it the gluten. In order to test the separation and identification of amino acids.
if the sample is free of starch, an Iodine
solution was used. A black solution 5. Qualitative Color Reactions
indicates a presence of starch while a For the qualitative analysis of
yellow solution is an indication that the proteins, separate test tubes were
sample is free of starch. Once the gluten prepared for each test, 10 of which
is free from starch, it can be subjected to contained an intact protein immersed in 1
qualitative analysis and hydrolysis. mL of distilled H2O. In another round of
test tubes, 0.5 mL of the hydrolyzed
2. Acid hydrolysis of intact protein sample was utilized for the same sample
First, 0.5 g of the isolated protein done in the procedure.
was placed in a test tube then 5 mL of 0.6
M HCl was added. The group labelled the A. Biuret Test
test tube with their year, section, course, The Biuret test was done by
and group number. The group also putting 20 drops of 2.5M NaOH to the
indicated that it was a H+ hydrolysis, and prepared sample. Afterwards, 2-3 drops of
included the name of the protein isolated.
0.1M CuSO4 solution was added to the
Next, the test tube was covered with
cotton and was handed to the professor
test tube. It was then mixed thoroughly. product was then recorded for the
The color reaction was later taken note of. presence of a red color reaction.

B. Ninhydrin Test G. Fohl’s Test

The Ninhydrin test made use of 6-10 The Fohl’s test was executed by
drops of 0.1% ninhydrin solution into the placing 5 drops of the 30% NaOH and 2
prepared test tubes. Right after, it was drops of the 5% Pb(CH3COO)2 to the
placed in a water bath to boil. The diluted samples. It was followed by the
samples were then observed for the submergence of test tubes to a boiling
presence of a blue violet color reaction. water bath. The presence of black or
brown sediments was then observed.
C. Xanthoproteic Test
The Xanthoproteic test was H. Test for Amides
performed by slowly placing 10 drops of The test for amides was carried out
concentrated HNO-3 to the diluted by adding 1mL of 20% NaOH to the test
samples for its initial step. After mixing tubes. Subsequently, it was placed in a
the test tubes, the color of the solution water bath to boil. While the prepared
was taken note of. Moreover, an addition samples are heating, a moistened red
of 10 drops of concentrated NaOH was litmus paper was put over the top of the
mixed to the samples. The resulting tube. A change in color of the litmus paper
solution was then recorded. was then noted.

D. Hopkins-Cole Test I. Pauly’s Test

The Hopkins-Cole test utilized 20 The Pauly’s test was done by mixing
drops of its reagent tso the diluted 3-5 drops of 1% sulfanilic acid and 3
samples. The inclined test tubes were drops of 5% NaNO2 solution for its diazo
added with 20 drops of concentrated reagent. Afterwards, 3-5 drops of 10%
H2SO4 along its edges. The color of its Na2CO3 was added to the mixture with the
interface was then observed. prepared sample. The resulting solution
was then observed for the presence of a
E. Sakaguchi Test red color reaction.
The Sakaguchi test was performed
by putting 10 drops of 10% NaOH and 10 RESULTS AND DISCUSSION
drops of 0.02% napthlol solution to the In this experiment, gluten is
prepared samples. After mixing the test isolated from the wheat flour by washing
tubes, it was left to stand for a maximum the dough with water. This process
removes the starch and the insoluble
of 3 minutes. It was then followed by
material found after is gluten [1]. After
adding 3 drops of 2% NaOBr to the obtaining the gluten, multiple qualitative
mixture. The resulting solution was then color reactions are performed to
taken note of. determine the different amino acids
present in the protein. Below this are the
F. Nitroprusside Test results and discussions of the data
gathered.
The Nitroprusside test was done by
mixing 0.5mL of 3M NaOH and 0.25mL of Table 1. Isolation of Proteins
2% nitroprusside solution. The resulting
Protein Description
 Gluten Dirty yellow gum-like
consistency

Casein White curd-like residue

Myoglobin Pale pink precipitate

Table 2. Hydrolysis of Intact Protein

Mode of Description of
Hydrolysis Hydrolysis
Figure 1.0. Intact Protein and Hydrolyzed
Acidic Protein after Biuret test
Acidic Brown solution
The Biuret test is utilized to detect
Basic Brown solution
the presence of peptide bonds. This test is
Enzymatic Clear solution, light based on the ability of the cupric ion from
orange cupric sulfate to form a violet/purple-
colored chelate complex from with peptide
bonds. The group was able to garner a
Qualitative Color Reactions
positive result with both intact and
The color tests have frequently
hydrolyzed protein in this test since both
been used for qualitative detection of
proteins gave off a lavender color. The
amino acids. Not all amino acids contain
hydrolyzed protein has its amino acid
the same reactive groups. These various
sequence broken down which makes it
color tests yield reactions varying in
easier for the cupric ion to form the
intensity and type of color according to
chelate complex giving it a more evidently
the nature of groups contained in the
colored shade of lavender.
particular amino acid under examination.
Table 4.1. Ninhydrin test for Intact Protein
Table 3.1. Biuret Test for Intact Protein
Color Reaction Intact Protein
Color Reaction Intact Protein

Ninhydrin Violet
Biuret Lavender

Table 4.2. Ninhydrin test for Hydrolyzed

Table 3.2. Biuret Test for Hydrolyzed Protein Protein

Color Hydrolyzed Protein Color Hydrolyzed Protein

Reaction Reaction

Acidic Basic Enzymatic Acidic Basic Enzymatic

Biuret Darker Lavender Light Ninhydrin Darker Dark Blue violet

lavend Purple violet violet
-er
Figure 2.0. Intact Protein and Hydrolyzed Figure 3.0. Intact Protein and
Acidic Protein after Ninhydrin test Hydrolyzed Acidic Protein after Xanthoproteic
test
The Ninhydrin test is a general and
typical test for an alpha-amino acid. The Xanthoproteic test is for
Ninhydrin is a powerful oxidizing reagent detection of aromatic side chains.
an in its presence, the amino acid Aromatic side chains usually exhibit
undergoes oxidative deamination, which is nitration. The nitric acid used in this test
the principle for this test, and this reacts with the aromatic side chain and
liberates ammonia, carbon dioxide, an produces the yellowish-colored solution.
aldehyde, and a reduced form of The principle involved in this test is
ninhydrin. The NH3 group of the amino nitration. All the results of the group had a
acid reacts with another molecule of positive result, with both intact and
ninhydrin and is reduced to give the blue hydrolyzed protein showing a yellow color.
substance, diketohydrin. The group
Table 6.1. Millon’s test for Intact Protein
managed to achieve a light purplish color
for the intact protein, and a successful Color Reaction Intact Protein
dark violet/blue color for the hydrolyzed
protein. The hydrolyzed protein for this
test also has a broken down amino acid
sequence, which removes other Millon’s Colorless
complications for the deamination to
succeed. [6]
Table 6.2. Millon’s test for Hydrolyzed Protein
Table 5.1. Xanthoproteic test for Intact Color Hydrolyzed Protein
Protein React-
ion
Color Reaction Intact Protein Acidic Basic Enzymatic

Millon’s Peach Cloudy White

White cloudy
Xanthoproteic Dark yellow

Table 5.2. Xanthoproteic test for Hydrolyzed

Protein
Color Hydrolyzed Protein
Reaction

Acidic Basic Enzymatic

Xantho- Yellow Pale Cream

proteic Yellow
Figure 4.0. Intact Protein and Hydrolyzed The Hopkins-Cole test is for
Acidic Protein after Millon’s test determination of tryptophan residues. The
indole group of tryptophan reacts with the
The Millon’s reagent reacts with a concentrated H2SO4 to give a purple-
hydroxybenzene ring (phenol group). The colored complex. The group’s results
only amino acid with a hydroxybenzene showed a positive result for the intact
ring is tyrosine, so this test is done to protein and the enzymatic hydrolyzed
detect the presence of tyrosine residues in protein with both proteins exhibiting a
specific proteins. Tyrosine when reacted purple interface. [4]
with acidified mercuric sulphate solution
gives yellow precipitate of mercury-amino Table 8.1. Sakaguchi test for Intact Protein
acid complex. On addition of sodium
Color Reaction Intact Protein
nitrate solution and heating, the yellow
complex of mercury-amino acid complex
converts to mercury phenolate which is in
red color. The results of the group were
far from the positive result with the intact Sakaguchi none
and hydrolyzed proteins showing no signs
of red pigments which means that gluten Table 8.2. Sakaguchi test for Hydrolyzed
does not have tyrosine residues. [5] Protein
Color Hydrolyzed Protein
Table 7.1. Hopkins-Cole test for Intact Protein
Reaction
Color Reaction Intact Protein
Acidic Basic Enzymatic

Sakaguchi none none none

Hopkins-Cole Purple interface
The Sakaguchi test is performed to
Table 7.2. Hopkins-Cole test for Hydrolyzed detect the presence of arginine in
Protein proteins. The principle of this test is the
Color Hydrolyzed Protein
complexation reaction. This is the reaction
Reaction of an amino acid containing guanido group
with alpha-Naphtol and sodium
Acidic Basic Enzymatic hypobromate. A positive result of this test
is a red or orange solution. Due to the
Hopkins- Less Colorless Purple unavailability of the reagents needed, the
Cole Cloudy group was not able to perform the test
White [4].
Table 9.1. Nitroprusside for Intact Protein
Color Reaction Intact Protein

Nitroprusside Yellow solution

Table 9.2. Nitroprusside for Hydrolyzed

Protein
Color Hydrolyzed Protein
Figure 5.0. Intact Protein and Hydrolyzed Reaction
Acidic Protein after Hopkins-Cole test
Acidic Basic Enzymatic
 Nitropru Orange Dark Yellow precipitat precipitat
sside solution yellow orange e e
solution

Figure 7.0. Intact Protein and Hydrolyzed

Figure 6.0. Intact Protein and Hydrolyzed Acidic Protein after Fohl’s test
Acidic Protein after Nitroprusside test
The Fohl’s test is performed to
The Nitroprusside test is performed detect the presence of sulfur containing
to detect the presence of cysteine, an amino acid. The principle behind this test
amino acid that contains a sulfhydryl is degradation and substitution reaction to
group. The principle of this test is from lead (II) sulfide. A positive indication
complexation reaction which causes the of this test is the formation of a black or
partial destruction of cysteine. Sodium brown precipitate from the lead (II)
nitroprusside reacts with compounds sulfide. Based on the table above, the
containing sulphahydryl groups. A positive color reaction of the intact protein, acidic
indication of this test is a red solution. hydrolyzed protein, basic hydrolyzed
Based on the table above, the color protein and enzymatic hydrolyzed protein
reaction did not result in a red solution, a all produced a positive result. This
possible reason behind this is indicates that sulfur is present in all of
contamination or an error in preparation. them. The intact protein is found to be
The closest color to the red solution is the darker compared to the hydrolyzed
result of the acid hydrolyzed protein which protein which means that the intact
produced an orange solution. The reason protein has more lead (II) sulfide [4].
behind this is that a hydrolyzed protein
solution has a greater plasma amino acid Table 11.1. Test for amides for Intact Protein
availability making it purer compared to Color Reaction Intact Protein
the isolated protein [4].

Table 10.1. Fohl’s test for Intact Protein

Color Reaction Intact Protein Test for Amides Yellow solution
Red - blue

Fohl’s Darker black precipitate Table 11.2. Test for amides for Hydrolyzed
Protein
Table 10.2. Fohl’s test for Hydrolyzed Protein
Color Hydrolyzed Protein
Color Hydrolyzed Protein Reactio
Reactio n
n Acidic Basic Enzymatic
Acidic Basic Enzymati
c
Test for Colorless Red- Clear
Fohl’s Black Brown Black Amides solution blue solution
 Red-blue litmus red-blue
paper

Figure 9.0. Intact Protein and

Hydrolyzed Acidic Protein after Pauly’s test

Figure 8.0. Intact Protein and Hydrolyzed The Pauly’s test is performed to
Acidic Protein after test for Amides detect the presence of tyrosine and
histidine. The principle behind this is the
The test for Amides is performed to formation of azo dyes where in tyrosine or
detect the presence of primary, histidine is coupled with diazonium salt in
secondary, tertiary, nitriles, asparagine alkaline condition. It involves the
and glutamine. The principle behind this is diazotization of sulfanilic acid in the
basic hydrolysis. A positive indication of presence of sodium nitrite and sodium
this test is the change of a red litmus nitrate. A positive indication of this test is
paper to blue. Based on the table above, a red solution. Based on the table above,
the change of red to blue litmus paper of the color reaction of the intact protein,
the intact protein, acidic hydrolyzed acidic hydrolyzed protein, basic
protein, basic hydrolyzed protein and hydrolyzed protein and enzymatic
enzymatic hydrolyzed protein all produced hydrolyzed protein all produced a positive
a positive result. The difference between result. This indicates the presence of
the intact protein and the hydrolyzed tyrosine and histidine. The protein that
protein cannot be seen since the change gave the strongest color is the acidic
of color of the litmus paper is the indicator hydrolyzed protein which produced a red
for the test [4]. orange solution. The reason behind this is
that a hydrolyzed protein solution has a
Table 12.1. Pauly’s test for Intact Protein
greater amino acid availability. This
Color Reaction Intact Protein means that it is purer compared to an
intact protein and will result in a stronger
color reaction [4].

Pauly’s Orange solution

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