Protein Isolation and Characterization

Tabaong, Mara Danielle; Tejada, Rolando; Ventura, Raymond; Villanueva, Marie

Antoinett; Villegas, Katrina; Vinluan, Kenna
Group 8 2DPH BIOCHEMISTRY LABORATORY

Abstract:

The intact protein was isolated from different sources by isoelectric precipitation,
difference in solubility and salt-induced precipitation. Casein was isolated from skimmed
milk, gluten from wheat flour and myoglobin from beef. The intact proteins were also
characterized by colorimetric reactions such as Biuret, Ninhydrin, Xanthoproteic,
Millon’s, Hopkins-Cole, Sakaguchi, Nitroprusside, Fohl’s, and Amide Tests. The Biuret
Test was done by adding 20 drops of 2.5 M NaOH and 2-3 drops of 0.1 M CuSO4
solution. The Ninhydrin Test was done by placing 6-10 drops of 0.1% ninhydrin solution
and by heating a boiling water bath. The Xanthoproteic Test was done by adding 10
drops of conc. HNO3 and 10 drops of conc. NaOH. The Millon’s Test was done by adding
5 drops of Millon’s reagent. The Hopkins-Cole Test was done by adding 20 drops of
Hopkins-Cole reagent and 20 drops conc. H2SO4. The Sakaguchi Test was done by
adding 10 drops each of 10% NaOH and 0.02% naphthol solution and 3 drops 2%
NaOBr. The Nitroprusside Test was done by adding 0.5 mL of 3 M NaOH and 0.25 mL
2% nitroprusside solution. The Fohl’s Test was done by adding 5 drops of 30% NaOH
and 2 drops of 5% (CH3COO)2Pb and placing into a boiling water bath. Lastly, the Test
for Amides was done by adding 1 mL of 20% NaOH and placing in water bath. All the
intact proteins showed a positive result: violet solution for Biuret Test, blue-violet
solution for Ninhydrin Test, orange solution for Xanthoproteic Test, flesh ppt. for Millon’s
Test, violet ring at the interface for Hopkins-Cole Test, red-orange solution for Sakaguchi
Test, yellow solution for Nitroprusside Test, black ppt. for Fohl’s Test, and red to blue
litmus paper and yellow-orange solution for Test for Amides.

Introduction: Structural proteins such as keratin and

A protein molecule is a long chain
of amino acids linked by peptide bonds.
The properties are determined by the
order or sequence of the amino acids in
its molecule, and by the three-
dimensional structure of the molecular
chain. The chain folds and twists and
then forming its conformational structure
which gives its distinctive properties [1].
Proteins are large, complex,
biologically-important molecules
composed of amino acids joined by
peptide bonds. The number of amino
acids used in a single protein can be
many hundreds. Proteins are essential to
all living organisms. As enzymes, they
regulate all aspects of metabolism.
collagen make up the skin, claws, bones,
tendons, and ligaments; muscle proteins
produce movement; hemoglobin
transports oxygen; and membrane
proteins regulate the movement of
substances into and out of cells. For
humans, protein is an essential part of
the diet, and is found in greatest
quantity in soy beans and other grain
legumes, meat, eggs, and cheese.
During digestion, protein molecules are
broken down into amino acids which are
then easily absorbed into the body [1].
Specific reactions are used for the
purpose of identifying amino acids and
proteins in biological media, for
qualitative and quantitative analysis.
Biuret test is used to determine peptide
bonds [2]. Ninhydrin test is typical for α-
amino acids [3]. Xanthroproteic test is a
test for the detection of aromatic
proteins in which concentrated nitric acid
reacts with the proteins to form a yellow
color that is intensified to orange-yellow
by the addition of alkali [4]. Millon’s test
is used to demonstrate the presence of
the amino acid tyrosine [5]. Hopkins-
Cole test is specific for tryptophan group
[6]. Sakaguchi test is a test for
guanidines, i.e arginine and peptides that
contain it [7]. Nitroprusside test is a test
for cystinuria. Fohl’s test is used to know
if sulfur-containing amino acids are
present [8]. Test for amides is used to
detect R-groups of asparagines and
glutamine.
The objectives of this experiment
are (1) to isolate casein from skimmed
milk by isoelectric precipitation, gluten
from wheat flour by difference in
solubility, and myoglobin from beef by
salt-induced precipitation; and (2) to
analyze chemical groups responsible for
color reactions and explain the principle
involved in each test.
Experimental:
Isolation of Casein from Skimmed Milk
Powdered non-fat milk with a
weight of 2.0 grams and 50.0 ml of
water was placed into a 100-ml beaker.
The mixture was then heated in a
temperature of about 400 C. A 10%
acetic acid was added dropwise when the
mixture has reached 400 C and then
stirred gently after every 5 drops. Acetic
acid was added continuously until the pH
reaches 4.6. The congealed casein was
filtered by using gravity filtration and
then the decantate was set aside for the
isolation of albumin. The casein residue
was dried then its weight % was
calculated.
Isolation of Gluten from Wheat Flour
Water was added to 1 cup of wheat
flour to make thick dough. The dough
was wrapped in the cheesecloth then
placed under running water until all
starch is removed. The washings was
tested with I2 solution until a negative
results is obtained. The insoluble
material was the crude gluten.
Isolation of Myoglobin from muscle
A 6.0 g minced beef was placed
with 6 ml 70 % (NH4)2SO4 solution in a
small beaker. The mixture was gently
stirred for 1 minute to release the
myoglobin. The dark-red extract was
expressed into a new beaker using
cheesecloth. The extract was centrifuged
at 13 000 x g for 15 minutes. A 1.5 ml of
supernatant was transferred into another
empty centrifuge tube. A ~0.30-0.35 of
(NH4)2SO4 crystals ground was added to
fine powder then mixed gently until the
solid dissolves. The samples were
centrifuged again for at least 5 minutes.
The supernatant was decanted off and
then the appearance of the purified
myoglobin residue was described.
Qualitative Color Reactions of the Intact
Proteins
The intact proteins were tested with
different characterization tests namely:
Biuret, Ninhydrin, Xanthoproteic,
Millon’s, Hopkins-Cole, Sakaguchi,
Nitroprusside, Fohl’s and Amide. There
were 9 test tubes prepared for each of
the test reaction. Each test tube
consisted of 0.5 g of intact protein in 1
mL distilled water. In Biuret Test, 20
drops of 2.5 M NaOH and 2-3 drops of
0.1 M CuSO4 solution were added to the
samples. Afterwards, they were shaken
and then observed for color changes. For
Ninhydrin test, 6-10 drops of 0.1 %
Ninhydrin solution was added to the
diluted sample and heated in a boiling because there are no net electrostatic
water bath. The appearance of a blue- repulsions between protein molecules. So
violet coloration was taken note of. In to isolate casein at its isoelectric pH, an
Xanthroproteic test, concentrated nitric acid is used to adjust the pH to 4.6.
acid and concentrated sodium hydroxide,
10 drops each, were added slowly and (b) gluten from wheat flour
then mixed. Color changes after each
addition were observed. In Millon’s test, Gluten was isolated from the wheat
5 drops of Millon’s reagent was added to flour by solubility differences. This was
the diluted sample with the color taken done by washing the dough with water.
note of. For Hopkins-Cole test, 20 drops This removes the starch; the insoluble
of Hopkins-Cole reagent was slowly material is the gluten.
added to the sample and mixed well. The
test tube was inclined to add slowly 20 (c) myoglobin from beef
drops of concentrated H2SO4. The color
at the interface was noted. In Sakaguchi Myoglobin was isolated from the
test, 10 drops each of 10% NaOH and beef by salt-induced precipitation
0.02 % α-naphthol solution was added to wherein the proteins are less soluble at
the samples then mixed and was let salt concentrations (high ionic strength)
stand for 3 minutes. Afterwards, 3 drops because the salt ions bind most of the
of 10% NaOBr was added. It was then water molecules. Myoglobin can be
mixed and color change was noted. In isolated by ammonium sulfate
Nitroprusside Test, 0.5 ml of 3M NaOH precipitation from the buffered muscle
and 0.25 ml 2 % nitroprusside solution extract.
was added. Formation of a red solution
was noted. In Fohl’s Test, 5 drops of 30 Qualitative Color Reactions of the Intact
% NaOH and 2 drops of 5% Proteins
(CH3COO)2Pb were added to the samples
then placed in a water bath. Appearance Table 1. Results obtained from the tests
of black precipitate was noted. Lastly, a Color Intact Protein
test for aide was made by adding 1 ml of Reaction (casein, gluten,
20 % NaOH to 10 drops of the sample myoglobin)
Biuret violet solution
then placed in a boiling water bath.
Ninhydrin blue-violet solution
Evolution of gas during heating was
Xanthoproteic orange solution
tested by placing a moistened red litmus
Millon’s flesh ppt.
paper over the mouth of the test tube Hopkins-Cole violet ring at the
and result was noted. interface
Sakaguchi red-orange solution
Results and Discussions: Nitroprusside yellow solution
Fohl’s black ppt
Isolation of Proteins Test for Amide red to blue litmus
paper; yellow-orange
(a) casein from skimmed milk solution

Casein was isolated from the
skimmed milk by isoelectric precipitation.
All proteins have an isoelectric point, pI,
a pH at which they have no net charge,
and they are least soluble at their pI
The Biuret Test for proteins
positively identifies the presence of
proteins in solution with violet color.
Biuret reacts with copper (II) ions in a
basic solution to form a violet complex.
The peptide linkages in proteins degradation and substitution reaction to
resemble those in biuret and also form form PbS.
deep violet complexes with basic copper
(II) ions in solution. The Test for Amide showed a
positive result of yellow-orange solution
The Ninhydrin Test showed a and the red litmus paper turned into
positive result of blue-violet solution. It blue.
is because proteins also contain free
amino groups on the alpha-carbon and
can react with ninhydrin to produce the
blue-violet color.

The Xanthoproteic Test showed a

positive result of orange solution. The
principle around this is the nitration of
aromatic rings via SEAR.
Figure 1. Colorimetric Results
The Millon’s Test showed a positive
result of flesh precipitate. In this test, References:
the phenol group of the tyrosine was
nitrated by nitric acid. The nitrated [1] Proteins.
tyrosine complexes mercury (I) and http://encyclopedia.farlex.com/Protein+(
mercury (II) ions into the solution to biochemistry). 8 January 2011. 5:15 pm.
form old rose/flesh to red precipitate. So
proteins with tyrosine will show a [2] Biuret Test.
positive result. http://www.scumdoctor.com/nutrition/pr
otein/Biuret-Test-For-Proteins.html. 8
The Hopkins-Cole Test showed a January 2011. 5:20 pm.
positive result of violet ring at the
interface. The indole ring reacts with [3] Ninhydrin Test.
glyoxylic acid in the presence of a strong http://www.chem.ucalgary.ca/courses/3
acid to form a violet ring product. 51/Carey5th/Ch27/ch27-3-3.html. 8
January 2011. 5:30 pm.
The Sakaguchi Test has a (+)
result of red to red-orange color. The [4] Xanthoproteic Test.
principle around this is about http://mw4.merriamwebster.com/medica
Complexation (base-catalyzed l/xanthoproteic%20test. 8 January 2011.
condensation of α-naphthol with the 5:34 pm.
guanido group of Arginine)
[5] Millon’s Test.
The Nitroprusside Test showed a http://botanydictionary.org/millons-
positive result of yellow solution because test.html. 8 January 2011. 5: 36 pm.
the cysteine group reacts with
nitroprusside in alkaline solution. [6] Hopkins-Cole Test.
http://www.cerlabs.com/experiments/10
The Fohl’s Test (Lead (II) acetate 875404480.pdf. 8 January 2011. 5:40
Test) has a (+) result of brown to black pm.
precipitate. The principle is about the
[7] Sakaguchi Test.
http://science.jrank.org/pages/41227/Sa
kaguchi-test.html#ixzz2AsWo5eEV. 8
January 2011. 6:00 pm.

[8] Nitroprusside Test.

http://www.mondofacto.com/facts/dictio
nary?nitroprusside+test. 8 January 2011.
6:04 pm.

[9] Fohl’s Test.

http://www.cerlabs.com/experiments/10
875404480.pdf. 8 January 2011. 5:41
pm.