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
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
Biochemistry Laboratory
• 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 α -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.
1
c. Xanthoproteic Test 3-5 drops of 10% Sodium Carbonate
In Xanthoproteic Test, the protein solution (Na2CO3) to the diazo reagent. Note the
is slowly treated with 10 drops appearance of the red coloration.
concentrated Nitric Acid (HNO3). Observe
caution for handling acids. Before
proceeding to the next step, take note of
the color of the solution. Next slowly add RESULTS AND DISCUSSION
10 drops of Sodium Hydroxide (NaOH) 1. Isolation of Gluten
and again note the color of the sample. Crude Gluten was obtained by washing the
dough to the water. The crude gluten obtained
d. Millon’s Test was from wheat flour, an ingredient to produce
The sample is treated with 5 drops of bread particularly leavened bread. Wheat flour
Millon’s reagent and be noted for its color has two major composition, starch and crude
reaction. gluten. In order to separate gluten from starch,
certain separation techniques must me used to
e. Hopkins-Cole Test separate the two components. The best way to
Slowly treat the gluten sample with 20 separate the two is by means of solubility.
drops Hopkins-cole reagent and mix well. Starch is a white odorless powdery
Then incline the tube add slowly along the carbohydrate (C6H10O5)x which is a chief storage
side 20 drops of concentrated sulfuric form of carbohydrates in plants [2]. Starch is
acid. (H2SO4). Observe caution. Note the soluble in water while gluten is insoluble. Hence
color of the sample. gluten can be separated by using the solubility
rule.
f. Sakaguchi Test Gluten is an elastic mixture composite of
The gluten sample is treated with 10% proteins glutenin and gliadin. Gluten has
Sodium Hydroxide (NaOH) and 10 drops chemical groups that make up its characteristics.
of 0.02% Naphthol Solution. Let the Gluten undergoes a series of Qualitative analysis
sample stand for 3 minutes and then add to further understand the structure of gluten.
3 drops 2% NaOBr. Again note the color
of the solution. 2. Qualitative Color Reaction of
Gluten
g. Nitroprusside Test In table 1, the results of the color reactions
For the nitroprusside test, add 0.5mL 3M are tabulated and noted. 0.5g of gluten was
of Sodium Hydroxide (NaOH) and 0.25 mL placed in ten test tubes together with 1mL of
2% Nitroprusside Solution. Note the Distilled water.
change in color to a red solution.
TABLE 1. RESULTS OF COLOR REACTION OF
h. Fohl’s Test GLUTEN
Add 5 drops of 30% Sodium Hydroxide COLOR REACTION TEST OBSERVATION/S
(NaOH) and 2 drops of Lead Acetate BIURET TEST Violet
(Pb(CH3COO)2) to the sample then NINHYDRIN TEST Blue Violet
proceed by placing it in a water bath. Note XANTHOPROTEIC TEST Yellow
for the appearance of dark or brown MILLON’S TEST White
sediments HOPKIN’S-COLE TEST Violet (interface)
SAKAGUCHI TEST Light Yellow
i. Test for Amides NITROPRUSSIDE TEST Red
Add 1mL 20% NaOH to 10 drops of the FOHL’S TEST Brown precipitate
TEST FOR AMIDES R-B (Basic)
gluten sample then proceed by placing it
PAULY TEST Pale Yellow
in the water bath. Test for the evolution of
gas by placing moisten red and blue
litmus paper over th mouth of the tube.
a. BIURET TEST (Test for Peptide
Bonds)
j. Pauly Test
First step is by preparing a diazo reagent The biuret test, it is basically the most
by mixing 3-5 drops 1% sulfosalicylic acid fundamental test for proteins, a general
with 3 drops of 5% Sodium Nitrite test for proteins [1]. The test is used for
(NaNO3). Add 5 drops of the sample and the presence of peptide bonds. When a
protein synthesizes with Copper (II)
2
 Sulfate, test will confirm a protein is
present in an unknown forming a protein-
copper complex which has a violet
coloration.
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)
3
 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.
From the Internet
[5] Undergraduate First Year Practical Manual
http://www.scribd.com/doc/183215/UNDERG
RADUATE-FIRST-YEAR-PRACTICALS-MANUAL

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. 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 α 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
4