Isolation, Qualitative Color Reaction and Alkaline Hydrolysis of Intact Protein Gluten

HAG Gutierrez, KL Hagiwara, CBDL Javier, AMF Labajo, AP Lansangan

Group 5, 2CMT, Faculty of Pharmacy, UST

Abstract
The experiment aims to isolate the intact protein which is gluten from wheat flour by difference in
solubility and to analyze the chemical group responsible for color reaction and explain the principle
involved in each test. Gluten is a protein composite that appears in foods processed from wheat and
related species, including barley and rye. It gives elasticity to dough, helping it to rise and to keep its
shape, and often giving the final product a chewy texture. The intact protein was isolated by washing the
dough with water which removes starch. Gluten was subjected for color reaction using the Biuret,
Ninhydrin, Xanthoproteic, Millon, Hopkins-Cole, Sakaguchi, Nitroprusside, Pauly, Fohl’s and Amide test
which determined the structure, functional group and presence of amide and aromatic side chains. The
intact protein was also alkaline hydrolyzed.

Introduction

Protein molecules are the builders of life; they
are essential constituent of all cells and most
abundant organic molecule found in living cells.
Proteins are polymers of amino acid monomer
units called residues which are bonded together
by peptide bonds. The precise order of amino
acid residues is known as the primary structure
of proteins. 4
Figure1. General structure of an amino acid
If the R- group contains a carboxyl group, the
amino acid displays acidic properties. When an
amino group is found instead, a basic amino
acid is the result and finally, alkyl groups
containing only carbon atoms or those with
alcohol or sulfur group for the R-group
constitute a neutral amino acid. In these
structures is an internal acid-base reaction can
occur as a resulting in formation of a
zwitterions, an internal salt, dipolar ion and is a
compound that contains both a positive and a
negative charge. 6
Figure2. Structure of a zwitterion
Proteins have precise three-dimensional
configuration, primary structure which refers to
its amino acid sequence which actually
determines the protein shape, secondary
structure which frequently contain α helix and
pleated sheet structures, tertiary structure
comprise of the further interaction of the
backbone of a protein, it is the folding and
coiling of a peptide chain to produce the
complex, rather rigid over-all conformation and
lastly, quaternary structure refers to the
interaction between subunits of a protein.
Those proteins that have only one subunit do
not possess a quaternary structure.
The “glue” that holds the shape together may
consist of hydrogen bonds, disulfide bonds,
hydrophobic interaction, ionic and covalent
bonds, electrostatic links and Van der Waals.
Many of these interactions take place between
side chain or R-groups of amino acid and
carboxyl group. 4
Hydrolysis of proteins refers to the breaking of
peptide bonds that link amino acids together.
The final result is the amino acid that makes up
the protein.4 The 20 amino acid commonly
found as hydrolysis product of protein contain
α-carboxyl group, an α-amino group and a
distinctive R-group substituted on a α-carbon
atom. It is done to obtain information about
their composition and can be carried out by
treating protein with acid, alkali, or proteolytic
enzymes.3
The protein that was isolated and was used to
test for determination of structure, functional
group, and aromatic and amide side chain is
gluten isolated from source which is wheat
flour.
Gluten can be defined as the rubbery mass that
remains when wheat dough is washed to
remove starch granules and water-soluble
constituents. It is a mixture of proteins not
readily soluble in water that occurs in wheat
and most other cereal grains. Its presence in
flour make production of leavened baked goods
possible because the chain-like gluten
molecules form elastic networks that traps
carbon dioxide gas and expands with it.
Materials and Method
A. Isolation of Gluten
In isolating Gluten, one cup of wheat flour,
cheesecloth and Iodine solution was required.
The one cup of wheat flour was added with
water until the dough turns thick. The dough
was wrapped in cheese cloth and was placed
under running water until all starch was
removed. The washings were tested for
presence of starch by adding Iodine solution
and were repeatedly done until a negative
result was obtained. The remaining insoluble
material in the cheesecloth is the crude gluten.
B. Qualitative Color Reaction of Gluten
For each test, a separate test tube with 0.5
grams of intact protein and 1 ml of distilled
water was prepared.
1. Biuret Test
In the prepared test tube with the intact
protein solution, 20 drops of 2.5 M NaOH
was added and mixed well. Another 2-3
drops of 0.1 M CuSO4 solution was added.
The test tube was shaken and the color of
the solution was noted.
2. Ninhydrin Test
The sample was treated with 6 – 10 drops
of 0.1% ninhydrin solution and was heated
in a boiling water bath. The color of the
solution was noted.
3. Xanthoproteic Test
In the prepared test tube with the intact
protein solution, 10 drops of concentrated
HNO3 was slowly added and was mixed. The
color of the solution was noted. Slowly
added after was 10 drops of concentrated
NaOH and was mixed well and the color of
the solution is again noted.
4. Millon’s Test
The sample was treated with 5 drops of
Millon’s reagent and the change in color
was noted.
5. Hopkins – Cole Test
For Hopkins-Cole test, 20 drops of Hopkins-
Cole reagent was slowly added to the
sample protein and was mixed well. The
test tube was inclined and 20 drops of
concentrated H2SO4 was slowly added along
the side. The color and the interface were
noted.
6. Sakaguchi Test
The sample was treated with 10 drops of
10% NaOH and 10 drops of 0.02% naphthol
solution and was mixed. It was left to stand
for 3 minutes, after, 3 drops of 2% NaOBr
was added and was mixed. The color
produced was noted.
7. Nitroprusside Test
In the prepared test tube with the intact
protein solution, 0.5 ml of 3 M NaOH was
added. Another 0.25 ml of 2% Nitroprusside
solution was placed. The decolorization of
the solution was noted.
8. Fohl’s Test
Five drops of 30% NaOH and 2 drops of 5%
(CH3COO)2 Pb was added to the intact
protein solution. After which, the test tube
 was placed in a boiling water bath and the conformation of the protein functional, its acid-
appearance of sediment was noted. base property and its solubility in proteins. 3

9. Test for Amides B. Qualitative Color reaction of Gluten

Ten drops of the protein sample solution Amino acids have a variety of chemically
was treated with 1ml of 20%NaOH. It was reactive groups that can be used to characterize
placed in a boiling water bath after. The both free amino acid and proteins. The
evolution of gas was tested during heating following tests are used to detect presence of
by placing a moistened red litmus paper amino acid and proteins and distinguish
over the mouth of the tube. The results between them.
were noted.
Table1. Results of Color Reaction of Intact
C. Alkaline Hydrolysis of Gluten Protein Solution
To hydrolyze the intact protein which is gluten, TEST COLOR REACTION
10 ml of 4 M NaOH was added to 0.5 g isolated Biuret Test Blue-Violet Solution
protein and was placed in a hard glass test tube Ninhydrin Test Blue-Violet Solution
and was labeled. The tube was covered with Xanthoproteic test Clear yellow Solution
cotton and was submitted for autoclaving in Millon’s Test Peach Solution
15psi for 5 hours after which the appearance Hopkins-Cole Test Clear Violet Ring
was noted and 10 ml of distilled water was Sakaguchi Test Clear yellow Solution
added and mixed well. The mixture was Nitroprusside Test Clear yellow Solution
transferred into a 250ml beaker. The mixture and brown sediments
was neutralized with 1M HCl and this Fohl’s Test Black precipitate
hydrolysate was used for another set of Test for Amides RedBlue Litmus
characterization test and chromatography. Paper
Results and Discussion 1. Biuret Test ( test for peptide bonds)
The Biuret test is a positive test for proteins but
A. Isolation of Gluten not for amino acid. The evidence for the test
consists of formation of a violet-pink complex
The isolated protein, Gluten was cream colored, when cupric ion, in basic solution is added to
sticky, elastic, gum-like texture and smells like any polymer such as protein which contains
of oatmeal. multiple amide bonds. A blue-colored solution
indicates a negative test or those fewer than
Wheat flour has two major components: crude two peptide bonds are present.3
gluten and starch. To separate the gluten which
is the intact protein from starch, a separation
technique is utilized, the best for which was
difference in solubility. Starch, a white odorless
powder carbohydrate which is a chief storage
form of carbohydrates in plant is soluble in
water while gluten, on the other hand, is an
elastic mixture composition of glutenins and
gliadins which is insoluble in water.
Figure3. Protein-Copper complex structure
Considered in the isolation of an intact protein formed in Biuret test. It is Blue violet in color.
from its sources are 3-Dimensional structure of
protein, the interaction that keeps the native
2. Ninhydrin Test ( test for α amino acid)
The ninhydrin test is positive for amino acid and
some proteins. 6 Free amino acids can react with
ninhydrin reagent yielding a deep purple
solution. Most of amino acids have free amino
groups Gluten is positive for this test.3

Figure6. Tyrosine (both free and constitutive of

proteins) reacts with reagent and produces a
purple-red salt of mercury

5. Hopkins-Cole Test (test for amino acid

Figure4. Two Ninhydrin molecules remove the α
tryptophan)
amino (NH2) group from an amino acid to form
The clear violet ring produced is due to the
a blue violet complex
formation of a compound from the glyoxylic
acid in the reagent and the tryptophan in the
3. Xanthoproteic test (test for aromatic side
protein. A similar color is produced when
chain)
sulphuric acid is added to a protein solution in
The test depends upon a reaction with a
the presence of a trace of formaldehyde. 7
specific type of amino acid chain. Aromatic rings
Gluten showed a positive result for the
often have the ability to undergo nitration
presence of tryptophan.
reaction, which is the addition of NO2 group to
the ring that is why it is a positive test for side
6. Sakaguchi Test (test for the guanidino
chains in tyrosine and tryptophan. With these
group of arginine)
amino acids present, addition of nitric acid and
In basic conditions, alpha naphthol and sodium
heat will result in a yellowish-colored solution.
hypobromite/chlorite react with the amino acid
Upon addition of base, color will change to
containing guanidine group to form red-orange
orange. Gluten is positive of aromatic side
complexes. Gluten sample showed negative
chain.
result thus, does not consist of arginine amino
acid.8

7. Nitroprusside Test (test for free –SH group)

Amino acid with free thiol groups due to
cysteine yields a positive result, red to red violet
Figure5. The Xanthoproteic test involves
decolorization when introduced to
replacing some of the hydrogen of the reactive
Nitroprusside. Gluten yields a positive result for
aromatic rings with nitro group forming a nitro
this test.9
compound pale yellow in color.
8. Fohl’s Test (test for sulfur containing
4. Millon’s Test (test for phenolic group
amino acid)
containing side chain)
This reaction is used for determination of S-
The color produced in Millon's test is given by
containing amino acids. Heating of protein
derivatives of benzene in which hydrogen in the
solution in an alkaline medium leads to the
ring has been replaced by a hydroxyl group. The
formation of sulfide (Na2S) if the protein
reaction serves as a test for the presence of
contained sulfur amino acids such as cysteine,
tyrosine.7 Gluten sample showed negative
cystine or methionine.8 A positive result is red
result.
to red violet decolorization. 9 Further reaction of
 5
Na2S will lead acetate a dark brown colored Copeland, R. (1994). Methods of protein
precipitate is formed8 Analysis. New York: Chapman and Hall. p 46

6
Peller, J. (1998). Exploring Chemistry:
Laboratory Experiments in General, Organic and
Biological Chemistry. Upper Saddle River NJ:
Prentice Hall. pp. 150 – 152

From websites:
7
Experimental Organic Chemistry. Retrieved on
Figure7. Fohl’s Reaction January 9, 2011 from:
http://www.books-about-
9. Test for Amides (test for the presence of california.com/Pages
Asparagine and Glutamine) /Experimental_Organic_Chemistry/Ex_Organic_
The red litmus paper turned blue indicates a Chem_Chap_26.html
basic component of the gluten, thus gluten is
8
positive for the presence of a basic amino acid. L.ivanovienė, R. Morkūnienė, R.Banienė. et al.
Laboratory Manual on Biochemistry. Retrieved
From the data gathered, Gluten was chemically January 9, 2011 from:
determined to be a basic amino acid contain http://www.kmu.lt/nsc/biochemija/Laboratory_
sulfide and peptide bonds, has an aromatic side manual_PART%20I.pdf
9
chain except for tyrosine. It is also positive for Vytheeshwaran Vedagiri. Undergraduate First
the presence of disulfide bond due to cysteine. Year Practical Manual. Retrieved on January 9,
2011 from:
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