Isolation and Characterization of Gluten from Wheat Flour

Abigail Beatrice H. Lumbao, Reham B. Macadato, Ryan B. Manzano,

Lina Dae T. Maranga and Eunice Nicole A. Martin
Group 4 2D Pharmacy Biochemistry Laboratory

ABSTRACT
Gluten was the protein found in wheat which remained when the wheat flour was processed into wheat
dough by addition of water and then was washed to take away starch granules as well as the water-
soluble constituents until such pure gluten was left. The presence of excess starch in the dough was tested
by addition of iodine solutions to the washings, wherein blue-violet coloration indicates such presence and
was washed again until a negative result was obtained. Gluten was then isolated, and situated in an
enzymatic substance for hydrolysis, which was then subjected to different qualitative tests.

INTRODUCTION Albumin is a protein made by the liver that

The class of compounds known as proteins is keeps fluid from leaking out of blood vessels,
an essential constituent of all cells. Proteins are nourishes tissues, and transports hormones,
copolymers of amino acids. There are 20 vitamins, drugs, and substances like calcium
naturally occurring amino acids namely Alanine, throughout the body.
Arginine, Asparagine, Aspartic acid, Cysteine, Gluten is a mixture of two proteins, glutenin
Glutamic acid, Glutamine, Glycine, Histidine, and gliadin. It is also the composite of a prolamin
Isoleucine, Leucine, Lysine, Methionine, and glutelin, which exist, conjoined with starch,
Phenylalanine, Proline, Serine, Threonine, in the endosperm of various grass-related grains.
Tryptophan, Tyrosine, and Valine that are Of the gluten proteins, gliadrin has a relative
commonly found in proteins. The sequence and molecular mass between 30000 and 80000 Da
organization of these amino acids contribute in whereas glutenin is multi-chained with relative
the unique physico-chemical characteristics of a molecular mass up to several million Da. It is
protein which determine what protein isolation along molecule having strong and flexible
and purification technique should be used. These characteristics. It is described as a yellowish-
characteristics include molecular structure, white, tough, elastic, and sticky protein. Given
molecular weight, solubility in different solvents, these characteristics, gluten became useful in
isoelectric pH, and heat stability. bread-making; for it traps the carbon dioxide
Isolation of proteins is the process for (CO2) produced by the reaction of flour and yeast
separating a type of protein from a complex and gives flour its characteristic chewiness,
mixture. Its importance is to characterize the helping it rise and keep its shape (Juacalla,
proteins solubility, acid-base property, function, 2011).
structure, and interactions. Proteins can be Myoglobin is a cytoplasmic hemoprotein,
isolated depending on their size, shape, charge, expressed solely in cardiac myocytes and
affinity, hydrophobicity, and other physiochemical oxidative skeletal muscle fibers, which reversibly
characteristics. binds O2 by its heme residue, a porphyrin ring:
Casein is a family of related phosphoproteins iron ion complex. Since the initial discovery of its
widely used as nutritional supplements in food structure over 40 years ago, wide-ranging work
and beverage. As a nutritional supplements, it by many investigators has added importantly to
can be used in a wide variety of industries our understanding of its function and regulation.
including: food production, beverage, Functionally, myoglobin is well accepted as an O 2-
pharmaceutical, and various other industries storage protein in muscle, capable of releasing O 2
(Incorporation, 2003). Casein is the most during periods of hypoxia or anoxia. Myoglobin is
abundant protein in milk. It is relatively insoluble also thought to buffer intracellular O2
and tends to form structures called micelles that concentration when muscle activity increases and
increase solubility in water. During the processing to facilitate intracellular O2 diffusion by providing
of milk, which usually involves heat or acid, the a parallel path that augments simple diffusion of
casein peptides and micelle structure become dissolved O2. The use of gene targeting and other
disturbed or denatured to form simpler molecular biological techniques has revealed
structures. As a result, a gelatinous material is important new insights into the developmental
formed. This is the basis for why casein has a and environmental regulation of myoglobin and
slower rate of digestion, and results in a slow but provided additional functions for this hemoprotein
steady release of amino acids into circulation. such as scavenging nitric oxide and reactive O2
species. These recent findings, coupled with and its appearance was
additional emerging technologies and the recorded,
discovery of other tissue globins, provide a b. Ninhydrin Test
framework for addressing new questions about 6-10 drops of 0.1% ninhydrin
myoglobin and readdressing old ones (George A. solution was placed into the
Ordway, 2004). sample. The sample was then
The objectives of this experiment are (1) subjected to a water bath. The
demonstrate commonly used protein isolation sample was observed if it gained
techniques and explain the principle involved in a violet color.
each isolation technique; (2) perform qualitative c. Xanthoproteic Test
tests on amino acids in intact and hydrolyzed 10 drops of concentrated
proteins and explain the principle involved in HNO3 was slowly added to the
each test; and (3) execute acid, alkaline and sample. The sample was mixed
enzymatic hydrolysis on the isolated proteins and and observed if there were any
enumerate the advantages and disadvantages of color changes. After that, 10
using each types of hydrolysis. drops of concentrated NaOH was
slowly added. Any color changes
EXPERIMENTAL were observed and recorded.
a. Test Compound/s (or Sample/s) d. Millons Test
used 5 drops of the Millons
A cup of wheat flour was used for the reagent was added to the diluted
isolation of the protein. sample. Any change in color was
b. Procedure noted.
e. Hopkins-Cole Test
1. Isolation of Protein (Gluten
20 drops of the Hopkins-Cole
from Wheat Flour) reagent was slowly added to the
A sufficient amount of water was sample. The test tube was
added to the cup of wheat flour to inclined and 20 drops of
make a thick dough. The dough was concentrated H2SO4 was added
washed with running water to remove along the sides of the test tube.
the starch in order to collect the The sample was not mixed. The
required protein. To test if the starch color at the interface was noted.
from the dough was already removed, f. Sakaguchi Test
the washings were tested with iodine 10 drops of 10% NaOH and
solution. The gluten was then collected 10 drops of the 0.02 naphthol
and was used for hydrolysis and were added to the samples. The
qualitative protein analysis. sample was left unattended for 3
2. Acid Hydolysis of Intact Protein minutes. After that, 3 drops of
5 mL of 6 M HCl was added to the the 2% NaOBr solution was
0.5 g isolated protein (gluten) in a added. The color produced was
hard glass test tube. The test tube was recorded.
then labeled. The test tube was then g. Nitroprusside Test
subjected for autoclaving for 5 hours 0.5 mL of 3 M NaOH was
with 15 psi. After autoclaving, the added to the 0.5 mL of the
appearance of the solution was sample. 0.25 mL of the 2%
observed. 10 mL was added to the nitroprusside solution was then
solution. The mixture was then added to the sample. The
transferred into a 250-mL beaker. The solution was the observed if it
mixture was neutralized with the use changed into a red solution.
of 1 M NaOH. The neutralized mixture h. Fohls Test
was then used for characterization 5 drops of the 30% NaOH
tests and chromatography. and 2 drops 5% Pb(CH 3COO)2
3. Qualitative Color Reactions were added to the sample. The
a. Biuret Test tube was then subjected to a
20 drops of 2.5 M NaOH was water bath. The appearance of
added to the sample and was black or brown sediments was
then mixed. Then, 2-3 drops of noted.
0.1 M CuSO4 was added to the i. Test for Amides
solution. The mixture was mixed
 1 mL of the 20% NaOH was
added to 10 drops of the sample. Figure1: Starch Iodine Complex
The sample was subjected to
water bath. The presence of gas
was tested using a litmus paper.
The red litmus paper was placed a. ACID HYDROLYSIS OF INTACT
over the mouth of the test tube. PROTEIN
The results were then noted. In acidic hydrolysis the isolated gluten must
j. Paulys Test hydrolyze by adding 5mL 6M HCl. The 6M HCl
The diazo reagent was serves as the catalyst to separate or denature the
preapared by mixing 3-5 drops gluten. After the mixture subjected to
of the 1% Sulfalinic acid with 3 autoclaving, the hydrosylate appeared to be a
drops of the 5% NaNO2 solution. black solution. Then, the mixture was neutralized
5 drops of the sample was added by 1 M NaOH to make as a sample for the
and 3-5 drops of 10% Na2CO3 characterization tests.
was added to the diazo reagent.
The appearance of a red b. QUALITATIVE COLOR REACTION
coloration was recorded. Amino acids have a variety of chemically
reactive groups. The reactions for side chains can
RESULTS AND DISCUSSION be used to characterize both free amino acids and
A. ISOLATION OF PROTEINS: GLUTEN proteins.
FROM WHEAT FLOUR
Wheat flour has two (2) major components: Table 1: Experimental results of qualitative
starch and gluten, and has a chemical formula color reaction
C6H10O5. COLOR
INTACT PROTEIN
Gluten refers to the proteins found in cereal REACTION
grains endosperm. It affects the elasticity of the Biuret Gray solution
dough, which in turn affects the chewiness of Ninhydrin Colorless
baked products. Gluten is actually composed of Xanthoproteic Colorless
two different proteins: gliadin and glutenin. Millons Flesh solution
Gliadin is a prolamin used chiefly as a nutrient in Hopkins-Coles Violet ring solution
high-protein diets and glutenin has an adhesive Sakaguchi Tan solution
property to flour. Starch is long chains of sugar Nitroprosside Dark yellow solution
molecules linked together like a chain. Provides Fohls Brown with sedimentation
more gradual energy source for bodily processes Test for amide Red to blue
than simple carbohydrates.. Starch is soluble Dark yellow with reddish
while gluten is insoluble. Hence the students can Pauly
shade
use the method of solubility to separate gluten
from the dough. c. Biuret Test
For this experiment, the students were Biuret test is often used to determine the
obtained gluten by washing the dough of wheat presence of peptide bonds in protein. It named
flour to the water. To check if the starch was after the substance biuret (H2NCONHCONH2). To
totally removed the students used a 0.01M iodine identify the results, see Table 2 below. Biuret
solution to the dough washings. For the positive reagents contains hydrated Copper sulphate,
result the washing will become deep blue in color. Potassium hydroxide solution, and Potassium
Amylose is one of the fraction of starch. It is the sodium tartrate. Hydrated Copper sulphate
responsible for the formation of a deep blue color provides the Cu (II) ions which form the chelate
in the presence of iodine. Because iodine slides complex. Cu (II) ions give the reagent its
into starch coil to give a blue-black color. (Figure characteristic blue color. Potassium hydroxide
1) For the negative result there is no color found. solution does not participate in the reaction but
provides the alkaline medium. And Potassium
sodium tartrate (KNaC4H4O64H2O) stabilizes the
chelate complex. A biuret test gives negative
result when a single amino acid (no peptide
bonds present - and dipeptides - only 1 peptide
bond present) is present. For this experiment, the
group obtained color gray solution. A gray in
color means there is an error or miscalculation in g. Hopkins-Cole Test
the experiment. Hopkins Cole test is a specific test used for the
detection of tryptophan. When tryptophan is
present in a solution, it gives a reddish violet
ring. When the violet ring appears after the two
Table 2: Expected results in the Biuret Test layers within an indole nucleus meet, this
for proteins. confirms that concentrated sulfuric acid was
OBSERVATION INTERPRETATION added to a mixture of some sort that contained
No change glyoxylic acid and a protein. However, there are
Proteins are not some products that do not show the reaction,
(solution remains
present such as gelatin and zein. The students observed a
blue )
The solution turns violet ring means a positive result.
from blue to violet Proteins are present h. Sakaguchi Test
(deep purple) Arginine and other guanidyl derivatives
Peptides are (glycocyamine, methylgyanidine etc) react with
present (Peptides or hypo bromide and alpha napthol to give a red
The solution turns colored product. The experiments sample give a
peptones are short
from blue to pink positive result.
chains of amino
acid residues) i. Nitroprusside Test
Sodium nitroprusside reacts with compounds
containing sulphahydryl groups produce an
d. Ninhydrin Test
intensely red but somewhat unstable color. It is
Ninhydrin Test (2,2dihydroxyindane-1,3-dione)
also a qualitative test for diagnosis of cystinuria;
is a test for amino acids and proteins with a free
the addition of fresh sodium cyanide formed by
-NH2 group. -amino-acid when warmed with
sodium nitroprusside to a sample of urine gives
triketohydrindene hydrate gave a fine blue color.
rise to a stable red-purple color in the presence
-amino- acids when substituted on the amino or
of cystine. The gluten sample give a yellow color
carboxyl group, gave a negative reaction.
means a negative result.
Proteins and their hydrolysis products were also
j. Fohls Test
found to give a positive reaction. To identify the
Fohls test is used to detecting sulfur-containing
result, see Table 3 below. For this experiment the
amino acids such as cysteine and cysteine. A
students observed that there is no color formed
positive result is red to red violet decolorization.
that means it is a negative result.
Further reaction of Na2S will lead acetate a dark
brown colored precipitate is formed. The sample
Table 3: Expected results in the Ninhudrin
give a positive result.
Test for proteins.
k. Test for amide
Observation Interpretation
Test for amide is test for the presence of
violet/purple product
Positive (+) Asparagine and Glutamine. The red litmus paper
forms
turned blue it means a basic component of the
no violet/purple product
Negative (-) gluten. From the students observed, Gluten was
formed
chemically determined to be a basic amino acid
contain sulfide and peptide bonds, has an
e. Xanthoproteic Test aromatic side chain except for tyrosine. It is also
Xanthoproteic test is used to determine the positive for the presence of disulfide bond due to
presence of tyrosine, trypthopane, and cysteine.
phenylalanine to the protein. In the presence of l. Pauly Test
concentrated nitric acid, the aromatic phenyl ring Pauly's test is a chemical test to detect the
is nitrated to give yellow colored nitro- amino acid histidine and tyrosine. Diazonium
derivatives. At alkaline pH, the color changes to component react with the imidazole ring of
orange due to the ionization of the phenolic histidine and a phenol group of tyrosine to form
group. (Amrita,2011) The students observed a dark red compound. Diazotisation is a reaction
colorless means a negative result. between an aromatic amine (in this case sulfanilic
f. Millons Test acid) with sodium nitrite and sodium carbonate to
A test developed by Auguste Millon, a French form diazonium component. Diazonium
chemist. This test is not specific for proteins. It component only form in cold conditions. The
detects phenolic compounds. A reddish-brown sample give a positive result.
coloration or precipitate indicates the presence of
tyrosine residue. The gluten sample give a
REFERENCES
positive result.
Dannie, M. (2015, April 30). What is the Function https://www.drugs.com/dict/cyanide-
of Starch? Retrieved March 19, 2017, from nitroprusside-test.html
Livesstrong.com:
http://www.livestrong.com/article/261545-list-of-
sugars-starches/ vlab.amrita.edu,. (2011). Qualitative Analysis of
Amino Acid. Retrieved 19 March 2017, from
Campbell, M., & Farrell, S. (2014). Supplement vlab.amrita.edu/?
(8th Edition ed.). Cengage Learning. sub=3&brch=63&sim=1094&cnt=1

Harding, V., & Warneford, F. The Ninhydrin Crisostomo, A.C., et. al. (2010). Laboratory
Reaction. McGill University. Manual in General Biochemistry. Quezon City: C
& E Publishing, Inc. Pages 18-25.
Kluwer, W. (2000). Cyanide-nitroprusside test.
Retrieved March 19, 2017, from Drugs.com: