Characterization of Intact Protein and Hydrolyzate by Color Reactions

C. Pallasigue, V. Pasia*, E. Rumbaoa, & C. San Diego

Group 8, 3Psy1, College of Science, UST

Keywords: Characterization, Color Reactions

Abstract
Casein, a protein that was isolated from milk from the previous experiment, half of it was
hydrolyzed and half of it was kept as an intact protein. The Intact Protein and Hydrolyzate were tested
using the Biuret test, Sakaguchi test, Ninhydrin test, Xanthoproteic test, and Hopkins-Cole test. Based on
the result, the Intact Protein (Protein suspension) is positive in the Biuret test, Sakaguchis test, and
Xanthroproteic test; while the Acid Hydrolyzate is positive in Ninhydrin test and Xanthroproteic test only.

Introduction
Amino acids are linked together by bonds called peptide linkages and hundreds or thousands of
these amino acids are bonded (called polypeptide chain) to from proteins. Most amino acids exhibit one
common feature: they have a primary amino group on the alpha-carbon; the carbon next to the carbonyl
group (Heasley, Christensen, & Heasley, 2013).The sequence of amino acid along the peptide chain is the
primary structure of a protein (Hein, Best, Miner, & Ritchey, 2001).
Proteins react with a variety of reagents to form colored products because of their constituent
peptide bonds and amino acids (Damodaran, 2011). The color produced in a given reagent varies because
the amount of amino acids that are present is different and/or the constituents that are attached to the
proteins are not much of a color-inducing group. Various substances other than proteins or amino acids
also give colors with some of the reagents (Hein, Best, Miner, & Ritchey, 2001). There were several tests
that was conducted to detect the presence of proteins in biological fluids or fluids with unknown
composition, such as the Biuret test and Ninhydrin test, as well as to identify the side chain groups of
amino acids, such as the Sakaguchis test, Xanthoproteic test, and Hopkins-Cole test.
The Biuret test involves heating a strongly alkaline solution of the test material with a little
copper(II) sulfate and is very sensitive to proteins and polypeptides containing at least three peptide units
(Hein, Best, Miner, & Ritchey, 2001). Ninhydrin reaction is chemical reaction to detect existence of amino
acids (The wall of Biochemistry, n.d.). The Xanthroproteic test is a characteristic reaction of proteins that
contain phenyl rings (Bettelheim & Landesberg, 2010). The Sakaguchis test shows whether a protein
solution contains amino acid with guanidino group, such as Arginine (Damodaran, 2011). The Hopkins-Cole
test determines the presence of the amino acid tryptophan (The wall of Biochemistry, n.d.).
There are several types or kinds of protein and one of it is Casein which was isolated from non-fat
milk from the previous experiment; half of which was turned into acid hydrolyzate and the other half being
the intact protein. Casein is released from its salts and precipitated milk from nonfat milk by treating with
dilute acetic or hydrochloric acids (Hein, Best, Miner, & Ritchey, 2001).This was the sample of protein that
was tested.

Methodology
Protein Suspension was prepared first by cutting the dried casein into small pieces and was placed
in a mortar. 10mL of distilled water was added and the Casein with distilled water was grinded until a fine
protein suspension was obtained. All of the tests for color reactions were done in medium sized test tubes,
the intact casein and the hydrolyzate were done side by side.
The first test that was done was the Biuret test. Three (3) drops of protein suspension was placed
in a test tube and three (3) drops of acid hydrolyzate was placed in another test tube. A drop of 2.5M NaOH
solution was added and mixed to each of the test tubes that contained the proteins, and then a drop of
0.01M CuSO4 solution was added and mixed. The color that was produced was noted.
The second test that was done was the Sakaguchi test in which five (5) drops of the protein
suspension was placed in test tube and five (5) drops of acid hydrolyzate was placed in another. A drop of
10% NaOH solution and a drop of 0.02% naphthol solution was added and mixed to each of the test tubes
containing the proteins. After about three (3) minutes, a drop of freshly-prepared 2% NaOBr was added and
the color produced was noted.
The third test that was done was the Ninhydrin test in which ten (10) drops of protein suspension
was mixed with 1mL of distilled water in a test tube while 1mL of the acid hydrolyzate was placed in another
without dilution with water. A 0.5mL of 0.1% of ninhydrin solution was added and mixed to each of the test
tubes containing the proteins, and was heated in a boiling water bath for 2-3 minutes. The color produced
was also noted.
The fourth test that was done was the Xanthoproteic test in which ten (10) drops of protein
suspension with 1mL distilled water was placed in a test tube while 1mL of acid hydrolyzate was placed in
another test tube without the dilution with water. Three (3) drops of concentrated HNO3 was slowly added
and mixed, and then the color produced was noted and then heated in boiling water bath for one (1)
minute. The solution was cooled down with flowing water and NaOH was added slowly drop by drop. The
addition of NaOH was continued until the solution is alkaline which was tested using the litmus paper. The
color produced was noted.
The fifth test was the Hopkins-Cole test in which 2 drops of protein suspension was placed in a test
tube while 2 drops of the acid hydrolyzate was placed in another test tube. 2mL of Hopkins-Cole reagent
was added and mixed well in each of the test tubes that contained the proteins. The test tubes were
inclined slowly and 2mL of concentrated H2SO4 was slowly added down the side of the tubes until two
layers were formed which was not disturbed. The color formed at the interphase was noted.

Results
Test Protein Suspension

Acid Hydrolyzate
Biuret Test After the addition of Sodium
hydroxide, the protein suspension
which was originally white in color
became transparent and clear
however it changed to a light
shade of violet after adding the
Copper (II) sulfate.

After the addition of Sodium
hydroxide, the color of the acid
hydrolyzate which was
transparent did not change
however, it turned into light shade
of blue upon adding Copper (II)
sulfate.
Sakaguchis Test After the addition of Sodium
hydroxide and the Napthol
solution, the protein suspension
became cloudy white and
changed into color red upon
adding Sodium hypobromite.

After adding the Sodium
Hydroxide, Napthol solution, and
Sodium hypobromite; the
hydrolyzate had two layers, the
top being transparent in color and
the bottom being yellowish in
color.

Ninhydrin Test After adding the Ninhydrin
solution it looked like it has white
precipitate, however after heating
it in the boiling bath, it turned into
dark shade of red.

There was no change in color
after adding the Ninhydrin
solution but after heating in the
boiling bath, it turned into violet.
Xanthoproteic Test After adding Nitric acid, there was
a white precipitate and after
heating it in the boiling bath, it
turned yellow with a dark yellow
precipitate.

After adding Nitric acid, it became
cloudy and after heating it in the
boiling bath, it became light
yellow.
Hopkins-Cole Test After adding the Hopkins-Cole
solution, it had two layers, the top
part being color white and the
bottom part being cloudy. After
adding the concentrated Sulfuric
acid, the top part continued to be
After adding the Hopkins-Cole
solution, it had two layers
however both of them looked
transparent in color. After adding
the concentrated Sulfuric acid,
the top part became faint color of
white while the bottom part
became transparent in color.

white while the bottom part
remained transparent in color.

Discussion
The Biuret test will yield positive results for proteins but not for amino acids. The evidence for the
test consist of the formation of a violet-pink complex when cupric ion, Cu
2+
, in basic solution was added to
any polymer such as protein, which contains multiple amide bonds (Heasley, Christensen, & Heasley,
2013). As for the results yielded a light shade of violet for the Protein Suspension, it shows that protein is
present; however for the Acid Hydrolyzate that yielded the light blue shade color, shows that protein is not
present.
Alpha-amino acids react with Ninhydrin (triketohydrindene hydrate) to form a blue to purple colored
complex (Hein, Best, Miner, & Ritchey, 2001). As for the results, the Protein suspension yielded a dark red
color which means that there are no Alpha-amino acids present while the Acid Hydrolyzate yielded a violet
color which means that there are Alpha-amino acids present in the solution.
In Sakaguchis test, alkaline pH guanidino group of arginine combines with alpha-napthol to form
bright red in color (Damodaran, 2011). As for the results from the experiment, the Protein suspension
yielded the color red which shows that guanidino group is present in the solution while the Acid hydrolyzate
yielded a transparent top and yellowish bottom layers which means that the guanidino group is not present.
In the Xanthroproteic test, concentrated nitric acid reacts with the phenyl ring to give a yellow-
colored aromatic nitro compound (Bettelheim & Landesberg, 2010). As for the results, both the Protein
suspension and the Acid hydrolyzate yielded the color yellow which means that in both of these solutions,
the phenyl ring is present.
The tryptophan that the Hopkins-Cole test determines is defined as an indole nucleus and is known
for creating the violet ring where the two layers meet (The wall of biochemistry, n.d.). In the protein
suspension and the acid hydrolyzate, the violet color where the layers meet was not present, which would
mean that tryptophan is not present in both of the solutions. However this is incorrect because the
tryptophan should have been present in the Protein suspension.

Conclusion
The Protein Suspension (Intact Protein) has protein that is present but no alpha-amino group; while
in the Acid Hydrolyzate, the protein is not present but the alpha-amino group is present. The guanidino
group is present for the protein suspension but not for the acid hydrolyzate. The phenyl ring is present to
both the protein suspension and the acid hydrolyzate however tryptophan is not present in both as based
on the result which is incorrect because tryptophan must be present in the protein suspension.
There are possible sources of errors; one is the human error of using the same dropper from a
solution to another. This may cause different reaction than the actual or rather correct reaction from the
experiment.
References
Bettelheim, F.A., & Landesberg, J.M. (2010). Laboratory Experiments for Introduction to General, Organic,
and Biochemistry (7
th
ed.). CA: Brooks/ Cole, Cengage Learning.
Damodaran, G.K. (2011). Practical Biochemistry. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd.
Heasley, V.L., Christensen, V.J., & Heasley, G.E. (2013). Chemistry and Life in the Laboratory:
Experiments (6
th
ed.). USA: Pearson Education Inc.
Hein, Best, Miner, & Ritchey. (2001). General, Organic, and Biochemistry in the Laboratory (7
th
ed.). USA:
Brooks/ Cole, Wadsworth Group.
The Wall of Biochemistry. (n.d.). Retrieved from http://fulltimes.wordpress.com/protein/