ISOLATION AND CHARACTERIZATION OF PROTEINS

(Basic Hydrolysis of Gluten)

Rio Pauline S. Roque, Deza Lyn M. Santos, Donna Rose J. Santos,

Gerald C. Sevilla and Celymar Angela M. Solis
Group 8 2A-Biochemistry Biochemistry Laboratory

ABSTRACT
Gluten is a protein found in wheat that remains when wheat dough is washed to remove starch granules and water-
soluble constituents. Gluten was isolated then hydrolyzed by base and was subjected to different qualitative tests.
Paper Chromatography was also performed to analyze the different amino acid components of gluten. Tests showed
that Gluten has several amino acid components.

INTRODUCTION tube which underwent autoclaving. 10ml of

Proteins, also known as polypeptides,
are organic compounds made of amino
acids arranged in a linear chain and folded into a
globular form. The amino acids in a polymer are
joined together by covalent bonds called peptide
bonds between the carboxyl and amino groups of
adjacent amino acid residues. The physical and
chemical properties unique to each amino acid
are the result of the structure and chemical
properties of the R group.
Hydrolysis is done to break peptide bonds, the
result of hydrolysis is smaller amino acid chains
or peptides, and free amino acids. The solution
containing the protein pieced is called a
hydrolysate solution. The protein to be
hydrolyzed in this experiment is Gluten, which
can be obtained from wheat flour. Certain color
reactions were done to determine the specific
amino acid components of Gluten.
Specific reactions are used for the purpose of
identifying amino acids and proteins in biological
media, for qualitative and quantitative analysis.
The Biuret test is used to detect the presence of
peptide bonds while the Ninhydrin reaction is a
typical test for an α-amino acid. Xanthoproteic
test detects side chains of aromatic amino acids
while the Millon’s and Hopkins-Cole tests
determine tyrosine and tryptophan residues,
respectively. The Nitroprusside test is used to
find out if sulfur-containing amino acids are
present; test for amides is used to detect R-
groups of asparagine and glutamine. Paper
Chromatography was also done to separate the
amino acid components of the protein gluten.
EXPERIMENTAL
HYDROLYSIS OF INTACT PROTEIN
After the protein Gluten was isolated, isolation
is not included in this formal report, it is then
hydrolyzed. Basic hydrolysis was done. 10ml of
4M NaoH was added to 0.5g of the isolated
protein, gluten. Cotton was used to plug the test
distilled water was added, the mixture was
transferred to a 250-ml beaker. 1M HCl was
added to neutralize the mixture.
QUALITATIVE COLOR REACTIONS
Biuret Test
20 drops of 2.5M NaOH was added to the
sample, it was mixed well. And then 2-3 drops of
0.1M CuSO4 solution was added. The mixture was
shook and the reaction was noted.
Ninhydrin Test
6-10 drops of 0.1% ninhydrin solution was
placed into the diluted sample. The tube was
heated in a boiling water bath. The appearance of
blue violet coloration was observed.
Xanthoproteic Test
10 drops of concentrated HNO 3 was slowly
added to the diluted sample, it was mixed and
the color was noted. 10 drops of concentrated
NaOH was added drop-wise, and again, the color
was noted.
Millon’s Test
5 drops of Milon’s reagent was added to the
diluted sample, and the color was observed.
Hopkins-Cole Test
20 drops of Hopkins-Cole reagent was added
drop-wise to the sample. With the test tube
inclined 20 drops of concentrated H2SO4 was
slowly added to the sample. The mixture was not
shoke to avoid violet reactions. The color at the
interface was observed.
Sakaguchi Test
10 drops of 10% NaOH and 10 drops of 0.02%
naphthol solution was added to the sample. It
was mixed and was left to stand for 3 minutes.
Then 3 drops of 2% NaOBr, it was mixed and the
color produced was noted.
Nitroprusside Test
0.5ml of 3M NaOH was added to an equal
amount of sample. Then 0.25ml 2% nitroprusside
solution was added. The formation of a red
solution was observed.
Fohl’s Test
 5 drops of 30% NaOH and 2 drops of 5% Color
(CH3COO)2Pb was added to the sample. The test Sakaguchi Flesh-Colored No Reaction
tube was placed in a boiling water bath. The sol’n
appearance of black or brown sediment was Nitroprusside Yellow sol’n Yellow sol’n
noted. Fohl’s Brown Brown
Test for Amides Sediment Sediment
1ml of 20% NaOH was added to 10 drops of Test for Red-Blue Red-Blue
the sample. The test tube was put in a boiling Amide Litmus Litmus
Pauly Colorless sol’n -----
water bath. A red litmus paper was placed on the
mouth of the test tube during heated that tested
for evolution of gas. The result was noted. Subjection of Gluten to base hydrolysis caused
Pauly Test the breakage of peptide bonds between the
A diazo reagent was prepared. 3-5 drops of amino acids.
1% sulfanilic acid was mixed with 3 drops of 5%
NaNO2 solution. 5 drops of the sample and 3-5 The Biuret test is used to test for the presence
drops of 10% Na2CO3 was added to the diazo of peptide bonds. Buiret solution contains Cu ions
reagent. The appearance of red coloration was which reacts with peptide bonds, thus, the intact
noted. protein yielded a positive reaction (purple
solution), and the completely hydrolyzed protein
SEPARATION AND IDENTIFICATION OF did not react.
AMINO ACIDS BY PAPER
CHROMATOGRAPHY Amino acids that have secondary amino group
A 5x12 cm filter paper was prepared. The attachments will react with Ninhydrin, producing
origin was drawn across the paper, 1.5cm from a yellow condensation product.
the bottom edge of the filter paper. 12
equidistant points were marked on the origin for Xanthoproteic Test yields a yellow product for
spotting of the amino acid standards, basic and positive aromatic ring containing amino acids.
acidic hydrolysate samples. The standards were The intensity of the yellow color deepens when
applied 5 times while the samples were applied the reaction occurs in a basic solution.
10 times with the use of capillary tubes. The filter
paper was placed inside the chamber with the Supposedly a positive result for Millon’s test is
solvent. The set-up was left to develop a red precipitate or red solution, but there are
undisturbed. The chromatogram was removed times that the reaction only yields a turbid or
from the chamber just as the solvent was 0.5cm white precipitate that turns red when heated.
from the top edge of the filter paper, the solvent Millon’s test is a test specific for tyrosine, the
was immediately marked. The chromatogram only amino acid containing a phenol group.
was air-dried, and sprayed with 1% ninhydrin
reagent. Then the chromatogram was place The Hopkins-Cole reagent only reacts with
above the hot plate. The spots were encircled proteins containing tryptophan. Tryptophan
and the Rf values were computed. reacts with glyoxylic acid to form the violet
product.

RESULTS AND DISCUSSION The Sakaguchi Test is used to detect the

presence of arginine. The sample was treated
with alpha-naphthol and sodium hypochlorite. A
Table 1. Results of the Qualitative Color
positive result yields a reddish wine color when
Reactions of the Intact Protein and the Base
arginine is present.
Hydrolysate.
The Nitroprusside Test is specific for cysteine,
Color Intact Basic
the only amino acid containing a sulfhydryl group
Reaction Protein Hydrolysate
(-SH). A red complex is yielded if sample
contains cysteine.
Biuret Purple sol’n Colorless sol’n
Ninhydrin Colorless sol’n Light Yellow Fohl’s test is used for determination of S-
sol’n containing amino acids. The reaction between
Xanthoproteic Dark Yellow Slightly sodium sulfide and lead acetate leads to the
Turbid sol’n formation of a dark brown precipitate.
Millon’s Colorless sol’n Turbid
Hopkins-Cole Violet Ring Light-Brown
 Test for amides was performed to test for the Cristobal, A.C., et al. 2010. Laboratory Manual in
presence of asparagine and glutamine amino General Biochemistry. Quezon City, Philippines:
acids in the sample. A positive result will show C&E Publishing Inc. pp.17-26
that litmus paper turns blue, an indication of
basic property. Voet, D.J., et al. 2008. Principles of Biochemistry.
River Street, Hoboken: John Wiley & Sons, Inc.
The basic principle involved in Pauly's test is pp.74, 312-313
diazotization. The sulphanilic acid gets diazotised
in the presence of sodium nitrite and sodium
carbonate with the sample. This test answers for Internet Based:
tyrosine, tryptophan and histidine residues. 
http://en.wikipedia.org/wiki/Gluten
Table 2. Rf values of the Amino Acid Standards
and the Basic Hydrolysate http://www.cerlabs.com/experiments/108754044
80.pdf
Amino Acid Rf Value
Standard Standards Basic http://www.chemguide.co.uk/analysis/chromatog
Hydrolysate raphy/paper.html
Tryptophan 0.57 0.57
Adenine 0.66
Proline 0.57 0.57
Cysteine 0.39 0.38
Serine 0.54
Aspartic acid 0.55
Tyrosine 0.65 0.63
Histidine 0.51 0.52
Glutamine 0.27 0.25
Alanine 0.37
*The values in the table has its discrepancy due
to errors done while doing the experiment.

Paper chromatography is a technique used to

separate different components of a certain
mixture. The principle behind this technique is
difference in polarity. The solvent system or
mobile phase is non-polar while the filter paper
or stationary phase is polar, thus components
that have higher Rf values are less polar than
components with lower Rf. The Rf value can be
computed with the use of this equation:

R distance travelled bycomponent

f=
distance travelled by solvent

Upon the results obtained, the conclusion is

that Tryptophan, Proline, Cysteine, Tyrosine,
Histidine, and Glutamine is present in the basic
hydrolysate of gluten. Due to multiple errors in
the procedure and analysis of the paper
chromatography, results may be incomplete and
may lack accuracy.

SOURCES

Book Based: