ISOLATION AND COLOR REACTION OF INTACT PROTEIN (GLUTEN)

Kriziaoumo P. Orpia, Michael Gavin G. Quinto, Nico Joy C. Ridulme

Kathryn Chemaine L. Samorano and Ryza Ruba
Group 8 2E Medical Technology Biochemistry Laboratory

ABSTRACT
Gluten, the intact protein, was separated from wheat flour by dissolving the starch that accompanied it in the process
of making wheat flour. The intact protein was tested to describe its structure and functional groups. Such test used
were to predetermine the presence for amides and aromatic side chains

INTRODUCTION
Gluten is a mixture of proteins not readily
soluble in water that occurs in wheat and most
other cereal grains. Its presence in flour makes
production of leavened baked goods possible
because the chain-like gluten molecules form
elastic network that traps carbon dioxide gas and
expands with it [4]. Gluten in the experiment is
taken from wheat flour. The objectives of this
experiment are to isolate gluten from the flour
mixture and to analyze and determine the
chemical group responsible for the color reactions
and explain the principle involved in each test.
EXPERIMENTAL
A. Materials and Compounds Used
1. For the Isolation of Gluten
 Wheat flour
 Water
 Cheesecloth
 Iodine Solution
2.For the Qualitative Color Reactions
 2.5M Sodium Hydroxide (NaOH)
 0.1M Copper Sulfate (CuSO4)
 Millon’s Reagent
 Hopkins-Cole Reagent
 10% Sodium Hydroxide (NaOH)
 0.1% Ninhydrin Solution
 3M Sodium Hydroxide (NaOH)
 2% Nitroprusside Solution
 30% Sodium Hydroxide (NaOH)
 1% Sulfosalicylic Acid
 10% Sodium Carbonate (Na2CO3)
 Conc. Sodium Hydroxide (NaOH) solution
 0.02% Naphthol Solution
 Conc. Nitric Acid (HNO3)
 Conc. Sulfuric Acid (H2SO4)
 6M Sodium Hydroxide (NaOH)
 2% NaOBr
 20% Sodium Hydroxide (NaOH)
 Hot plate
 Beaker
 5% Lead Acetate (Pb(CH3COO)2)
a. 5% Sodium Nitrite (NaNO2)
B. Procedure
1. Isolation of Gluten
In isolating gluten, one cup of wheat flour is
washed with water. Continue to add water until
the dough turns thick. As the dough thickens,
wrap it onto the cheesecloth and place it in
running water. This way starch is removed from
the dough. The procedure is done until such time
all the starch from the dough is removed. To test
whether starch is still present, gather water
droppings below the dough in a test tube and use
iodine solution. If the iodine solution is negative,
it can be safe to assume that all starch is
removed from the dough. The remaining
insoluble material inside the cheesecloth is your
crude gluten.
2. Qualitative Color Reaction of Gluten
The test here aids to identify the chemical
groups that are bonded to the -carbon. In ten
test tubes, 0.5g of gluten is dissolved in 1 mL
distilled water. Each test tube corresponds to a
particular test. Label each test tube according to
their corresponding test to avoid confusion.
a. Biuret Test
For the biuret test, treat the protein
solution with 20 drops of 2.5M NaOH and
mix well. Next step is by adding 2-3 drops
of 0.1M CuSO4 solution. Take note of the
color of the gluten solution after treated.
b. Ninhydrin Test
For the Ninhyrin Test, treat the diluted
sample with 6-10 drops of 0.1% Ninhydrin
solution then proceed by placing the test
tube containing the sample in a water bath
and observe the appearance of a blue violet
coloration.
c. Xanthoproteic Test
In Xanthoproteic Test, the protein solution
is slowly treated with 10 drops
concentrated Nitric Acid (HNO3). Observe

1
 caution for handling acids. Before
proceeding to the next step, take note of RESULTS AND DISCUSSION
the color of the solution. Next slowly add 1. Isolation of Gluten
10 drops of Sodium Hydroxide (NaOH) and Crude Gluten was obtained by washing the
again note the color of the sample. dough to the water. The crude gluten obtained was
from wheat flour, an ingredient to produce bread
d. Millon’s Test particularly leavened bread. Wheat flour has two
The sample is treated with 5 drops of major composition, starch and crude gluten. In
Millon’s reagent and be noted for its color order to separate gluten from starch, certain
reaction. separation techniques must me used to separate
the two components. The best way to separate the
e. Hopkins-Cole Test two is by means of solubility.
Slowly treat the gluten sample with 20 Starch is a white odorless powdery
drops Hopkins-cole reagent and mix well. carbohydrate (C6H10O5)x which is a chief storage
Then incline the tube add slowly along the form of carbohydrates in plants [2]. Starch is
side 20 drops of concentrated sulfuric acid. soluble in water while gluten is insoluble. Hence
(H2SO4). Observe caution. Note the color of gluten can be separated by using the solubility
the sample. rule.
Gluten is an elastic mixture composite of
f. Sakaguchi Test proteins glutenin and gliadin. Gluten has chemical
The gluten sample is treated with 10% groups that make up its characteristics. Gluten
Sodium Hydroxide (NaOH) and 10 drops of undergoes a series of Qualitative analysis to
0.02% Naphthol Solution. Let the sample further understand the structure of gluten.
stand for 3 minutes and then add 3 drops
2% NaOBr. Again note the color of the 2. Qualitative Color Reaction of Gluten
solution. In table 1, the results of the color reactions are
tabulated and noted. 0.5g of gluten was placed in
g. Nitroprusside Test ten test tubes together with 1mL of Distilled water.
For the nitroprusside test, add 0.5mL 3M of
Sodium Hydroxide (NaOH) and 0.25 mL TABLE 1. RESULTS OF COLOR REACTION OF
2% Nitroprusside Solution. Note the GLUTEN
change in color to a red solution. COLOR REACTION
OBSERVATION/S
TEST
h. Fohl’s Test BIURET TEST Violet
Add 5 drops of 30% Sodium Hydroxide NINHYDRIN TEST Blue Violet
(NaOH) and 2 drops of Lead Acetate XANTHOPROTEIC TEST Yellow
(Pb(CH3COO)2) to the sample then proceed MILLON’S TEST White
by placing it in a water bath. Note for the HOPKIN’S-COLE TEST Violet (interface)
appearance of dark or brown sediments SAKAGUCHI TEST Light Yellow
NITROPRUSSIDE TEST Red
FOHL’S TEST Brown precipitate
i. Test for Amides
TEST FOR AMIDES R-B (Basic)
Add 1mL 20% NaOH to 10 drops of the
PAULY TEST Pale Yellow
gluten sample then proceed by placing it in
the water bath. Test for the evolution of a. BIURET TEST (Test for Peptide Bonds)
gas by placing moisten red and blue litmus
paper over th mouth of the tube. The biuret test, it is basically the most
fundamental test for proteins, a general
j. Pauly Test test for proteins [1]. The test is used for
First step is by preparing a diazo reagent the presence of peptide bonds. When a
by mixing 3-5 drops 1% sulfosalicylic acid protein synthesizes with Copper (II)
with 3 drops of 5% Sodium Nitrite (NaNO3). Sulfate, test will confirm a protein is
Add 5 drops of the sample and 3-5 drops of present in an unknown forming a protein-
10% Sodium Carbonate (Na2CO3) to the copper complex which has a violet
diazo reagent. Note the appearance of the coloration.
red coloration.

2

Illustration from :
Laboratory Experiments
for Organic and
Biochemistry
b. NINHYDRIN TEST (test for Free NH2)
Amino acids with a free –NH2 group and
proteins containing free amino groups
react with ninhydrin to give a purple-blue
complex. Gluten resulted positive to the
test.
Illustration from: Laboratory Experiments for
Organic and Biochemistry
c. XANTHOPROTEIC TEST (test for
Aromatic Side Chains)
The Gluten sample reacted to the
concentrated Nitric Acid (HNO3) forming a
yellow substance or aromatic nitro
compound [5]. After adding an alkali or a
strong base in the form of Sodium
Hydroxide, the color shifted to orange.
Such amino acids like tyrosine,
phenylalanine and tryphtophan. The gluten
sample is positive for aromatic side chains.
d. MILLON’S TEST (test for phenolic
group containing amino acids)
The Millon’s test is the test to determine
the presence of Tyrosine [5], which is the
amino acid that contains a phenolic ring,
but the gluten sample which was tested for
this test showed negative result. The
gluten contains side aromatic chains except
tyrosine.
e. HOPKINS-COLE TEST (test for the
amino acid tryphtophan)
The gluten sample was first treated with
Hopkins-Cole reagent and then slowly
added concentrated Sulfuric Acid (H2SO4).
The visible result would be the color at the
interface of the two substances. The
interface is colored violet. The positive
result of Hopkins-Cole test is a violet color.
This violet color is due to the presence of
the indol group. [5] Gluten is positive with
tryptophan
f. SAKAGUCHI TEST (Test for the
guanidino group of Arginine)
The Sakaguchi test is a test for the
presence of the guanidine group
specifically found in Arginine [5]. The
gluten sample however test for a negative
results which generally means the gluten
sample does not consist if a Arginine amino
acid.
g. NITROPRUSSIDE TEST (test for free –
SH group)
The test results to a red color or red violet.
Such result can be seen in Cysteine.
Therefore it can be stated that the amino
acids with free thiol groups due to cystiene
yields a red color when introduced to
Nitroprusside’s or Bollin’s Test [5]. Gluten
was positive for the results.
h. FOHL’S TEST (test for sulfur containing
amino acids)
The Fohl’s test shown gluten was positive
for the brown precipitate. Fohl’s test is the
same in test as to nitroprusside test. They
are the test to determine is the protein
contains sulfur bound compounds.
i. TEST FOR AMIDES (test for the
presence of Asparagine and
Glutamine)
The gluten solution was treated with
Sodium Hydroxide and was hot bathed until
evolution of gas occurs. Using litmus paper,
the red litmus paper turned blue. This
indicates a basic component of gluten.
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 Gluten is positive for the presence of basic
Amino Acids

j. PAULY TEST (Test for Histidine and

Tyrosine)

The gluten sample showed negative result

to the Pauly test and denies the presence
of Histidine. The diazo Reagent would react
to the presence of amino immidazole and
phenol to form a colourful azo compound
(deep red) [5].

The gluten in general is a mixture of protein

consisting of glutenin and gliadin. From the results
gathered from the color reaction, Gluten can be
chemical described as an amino acid that contains
sulfide bonds, peptide bonds. Some chains
between each  carbon are also examined. Gluten
has an aromatic side chain except from tyrosine.
Gluten was positive for the presence of basic
amino acids. It was also positive for the presence
of disulfide bond due to cysteine.

REFERENCES

From Books
[1] Bettelheim, Frederick A., Landersberg
Joseph M. (2001), Laboratory Experiments
for Organic and Biochemistry Philadelphia:
Harccourt College Publishers. P.201
[2] Merriam-Webster’s Medical Dictionary
(2006). Springfield Massachusetts:
Merriam-Webster Incorporated
[3] Copeland, Robert.(1994). Methods of
Protein Analysis NewYork: Chapman &
Hall,p.46
[4] Gluten, Britannica Concise Encyclopedia
(2002) U.S.A.: Encyclopædia Britannica

From the Internet

[5] Undergraduate First Year Practical Manual

http://www.scribd.com/doc/183215/UNDERG
RADUATE-FIRST-YEAR-PRACTICALS-MANUAL