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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.
Isolation of Casein from Skimmed Milk Qualitative Color Reactions of the Intact
Proteins
Powdered non-fat milk with a
weight of 2.0 grams and 50.0 ml of The intact proteins were tested with
water was placed into a 100-ml beaker. different characterization tests namely:
The mixture was then heated in a Biuret, Ninhydrin, Xanthoproteic,
temperature of about 400 C. A 10% Millon’s, Hopkins-Cole, Sakaguchi,
acetic acid was added dropwise when the Nitroprusside, Fohl’s and Amide. There
mixture has reached 400 C and then were 9 test tubes prepared for each of
stirred gently after every 5 drops. Acetic the test reaction. Each test tube
acid was added continuously until the pH consisted of 0.5 g of intact protein in 1
reaches 4.6. The congealed casein was mL distilled water. In Biuret Test, 20
filtered by using gravity filtration and drops of 2.5 M NaOH and 2-3 drops of
then the decantate was set aside for the 0.1 M CuSO4 solution were added to the
isolation of albumin. The casein residue samples. Afterwards, they were shaken
was dried then its weight % was and then observed for color changes. For
calculated. 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 The Biuret Test for proteins
skimmed milk by isoelectric precipitation. positively identifies the presence of
All proteins have an isoelectric point, pI, proteins in solution with violet color.
a pH at which they have no net charge, Biuret reacts with copper (II) ions in a
and they are least soluble at their pI 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.