PH-BIOCHEM Expt 2 2FPH Group 1

QUALITATIVE COLOR REACTIONS OF INTACT AND HYDROLYZED CASEIN

FROM NON-FAT MILK
Alonzo, C.A., Banta, C.J., Cabais, M.J.A., Cabonegro, A.M., Cortez, J.F.*
Department of Pharmacy, Faculty of Pharmacy
University of Santo Tomas

ABSTRACT
This experiment used non-fat milk as the source of the protein isolate, casein. After
isolation and hydrolysis of the protein, qualitative tests were performed on both the protein
isolate and hydrolysate. The experiment aims to enable the students to perform qualitative
tests and explain the principles involved in each of the test. Out of the 10 qualitative tests,
the isolate tested positive for the following tests: Biuret, Ninhydrin, Xanthoproteic,
Sakaguchi, Fohls, Test for Amides, and Paulys. On the other hand, the hydrolysate tested
positive for the following tests: Ninhydrin, Xanthoproteic, Fohls, Test for Amides, and
Paulys. This indicates that acid hydrolysis may have destroyed or affected some of the
amino acid components of casein. Thus, qualitative tests alone are insufficient in identifying
the amino acid components of a protein. Factors such as cross-contamination of reagents
and the effect of acid hydrolysis are some sources of errors that may render the tests
inaccurate.

INTRODUCTION calcium caseinate in milk.[7]

Proteins in structure are made
up of hundreds or more amino acids
bind together with peptide bonds. [3]
They are essential for the function and
regulation of the bodys tissues and
organs. Proteins may be classified
according to their function.[9] In the
case of casein, it is classified as a
storage protein.

Milk is mixture of various types

of proteins and it is a primary food
Figure 1. Casein micelle
source for infant mammals. Even
though mammals stop drinking milk The objective of this experiment
after maturity, it is used in the is to perform qualitative tests on the
production of various dairy products amino acids in intact and hydrolyzed
such as cheese, butter, and cream proteins and explain the principles
which are then used in cooking and involved in each test.
baking. Milk contains three main
proteins: casein, lactalbumin, and METHODOLOGY
lactoglobulin.[2] Casein is the A. Qualitative Color Reactions of
predominant protein found in milk Casein
about 80% of the protein component
of milk. It is a heterogenous mixture of Dissolve a pinch amount of
phosphorus containing proteins (alpha, intact protein in 1 mL distilled
beta, and kappa casein) present as water. Place 0.5 mL of
PH-BIOCHEM Expt 2 2FPH Group 1
2
hydrolyzed sample in separate
test tubes.
Figure 1. Preparation of intact
protein solution
1. Biuret Test
In the prepared test tube
with intact protein solution,
20 drops of 2.5 M NaOH and
2-3 drops of 0.1 M CuSO 4
were added.
2. Ninhydrin Test
6-10 drops of 0.1%
ninhydrin solution was
placed in the test tube. Place
test tube in a boiling water
bath.
3. Xanthoproteic Test
10 drops each of conc.
HNO3 and conc. NaOH were
mixed with the samples.
4. Millons Test
5 drops of the Millons
reagent were dropped into
the prepared test tubes.
5. Hopkins-Cole Test
20 drops of the Hopkins-
Cole reagent was added and
mixed into the test tubes.
The test tube was positioned
in a diagonal manner and 20
drops of conc. H2SO4 were
dropped at the sides of the
test tubes.
6. Sakaguchi Test
10 drops each of 10%
NaOH and 0.02% naphthol
solution were added and
mixed into the test tubes.
The mixture was left to stand
for 3 minutes, after which 3
drops of 2% NaOBr was
added and mixed.
7. Nitroprusside Test
10 drops of 3 M NaOH
and 5 drops of 2%
nitroprusside solution were
added to the test tubes.
8. Fohls Test
5 drops of 30% NaOH and
2 drops of 5% Pb(CH3COO)2
were added to the test
tubes. The test tubes were
then placed in a boiling
water bath.
9. Test for Amides
20 drops of 20% NaOH
were added to the test
tubes. The test tubes were
then placed in a boiling
water bath. A moistened red
litmus paper was placed at
the mouth of each test tubes
to check for evolution of gas.
10.Paulys Test
5 drops of 1% sulfanilic
acid and 3 drops of 5%
PH-BIOCHEM Expt 2 2FPH Group 1
3

NaNO2 solution were mixed Test for (+) yellow (+) dirty
into each test tubes. 5 drops Amide soln yellow soln
of 10% Na2CO3 were then RB RB
added. litmus litmus
Paulys (+) orange (+) red soln
soln
RESULTS AND DISCUSSION
Table 1 below shows the results
Biuret Test is a tests for the
of the qualitative tests of intact casein
presence of peptide bonds. The
and hydrolyzed casein. These tests
principle involved is the formation of
help identify the amino acid
coordination complexes with copper
constituents of the protein through
(II). Because hydrolysis destroys the
color reactions with the different
primary structure of proteins, peptide
functional groups.
bonds are not present in the
hydrolysate.[6] Thus, the intact protein
Table 1. Qualitative Color Reactions of
gives a positive result while the
Intact Casein and Hydrolyzed Casein
hydrolysate gives a negative result.
Color Intact Acid
Reaction Protein Hydrolysis
Biuret (+) purple (-) black
soln soln
& ppt
Ninhydrin (+) violet (+) dark
ppt violet
soln & ppt Figure 1. Coordination complex
Xanthoprot (+) yellow (+) orange formation of copper (II)
eic ppt soln
& soln
Millons (-) white ppt (-) brown
soln
Hopkins- (-) white ppt (-) brown &
Cole colorless
layered soln
w/ brown
ring
Sakaguchi (+) light (-) brown
yellow soln ppt and
soln
Nitroprussid (-) yellow (-) yellow
e soln soln w/
brown ppt
Fohls (+) (+) brown
brownish ppt & soln Figure 2. Biuret Test on intact protein
black soln & (left) and hydrolysate (right)
ppt
PH-BIOCHEM Expt 2 2FPH Group 1
4

Ninhydrin Test is a test for the Figure 5. Nitration of phenyl group

presence of alpha-amino groups. The
principle involved is the oxidative
deamination of alpha-amino groups.
The positive result of this test is blue-
violet solution and precipitate. Both
intact protein and hydrolysate tested
positive for this reaction.

Figure 3. Oxidative deamination of

alpha-amino group

Figure 6. Xanthoproteic Test on intact

protein (left) and hydrolysate (right)

Millons Test is a test for the

presence of phenolic compounds such
as tyrosine. The principle involved is
the reaction of phenol with mercuric
nitrate producing red precipitate which
then reacts with the nitric acid in the
solution forming a red solution. The
positive result of this test is old rose or
red precipitate.[6] Both the intact
Figure 4. Ninhydrin Test on intact protein and hydrolysate should have a
protein (left) and hydrolysate (right) positive result. However, in the
experiment, both the intact protein
Xanthoproteic is a test for the and hydrolysate gave a negative
presence of aromatic groups such as result.
tyrosine, tryptophan, and
phenylalanine. But phenylalanine, due
to its inactivity, does not show a
positive result. The principle involved
is the nitration of the phenyl group,
C6H5. A positive result of this test is a
yellow solution.[6] Both intact protein
and hydrolysate showed a positive
result for this test, indicating that
casein contains an aromatic group.
PH-BIOCHEM Expt 2 2FPH Group 1
5

oxidation of the guanidine group by

hypo bromide and alpha napthol
producing a red solution.[6] Both intact
protein and hydrolysate should have a
positive result. However, only the
intact protein produced a light yellow
solution in this reaction while the
hydrolysate remained the same color.

Figure 7. Millons Test on intact protein

(left) and hydrolysate (right)

Hopkins-Cole Test is a test for

the presence of indole group found in
tryptophan. The positive result for this
test is a violet ring formed due to the
reaction of the indole group with
gyloxylic acid in the presence of a
strong acid.[6] According to Table 1, Figure 9. Sakaguchi Test on intact
both the intact protein and protein (left) and hydrolysate (right)
hydrolysate showed a negative result.
Nitroprusside Test is a test for
the presence of sulphahydryl groups
such as cysteine. The principle
involved is the complexation reaction
of the sulphahydryl groups with
sodium nitroprusside producing a red
solution.[6] In the experiment, both
intact protein and hydrolysate
produced a yellow solution. This is due
to only small amounts of cysteine
found in casein.[8]

Figure 8. Hopkins-Cole Test on intact

protein (right) and hydrolysate (left)

Sakaguchi Test is a test for the

presence of guanidine group such as
arginine. The principle involved is the
PH-BIOCHEM Expt 2 2FPH Group 1
6

hydrolysis of amines. In alkaline

conditions, amides give off ammonia
upon heating, turning red litmus paper
to blue.[4] Both intact protein and
hydrolysate produced a positive result
in this test.

Figure 10. Nitroprusside Test on intact

protein (left) and hydrolysate (right)

Fohls Test is a test for the

presence of sulfur-containing amino
acids such as cysteine and Figure 12. Test for Amides on intact
methionine. The principle involved is protein (left) and hydrolysate (right)
the reaction of sodium sulfide with Paulys Test is a test for the
lead acetate to give lead sulfide (PbS), presence tyrosine and histidine. The
a brown to black precipitate. [8] Both principle involved is the reaction of the
intact protein and hydrolysate imidazole ring of histidine and phenol
produced a positive result in this test. group of tyrosine with the diazonium
component, forming a red solution. [5]
Both intact protein and hydrolysate
produced a positive result.

Figure 11. Fohls Test on intact protein

(left) and hydrolysate (right)

Test for Amides is a test for the

presence of asparagine and glutamine. Figure 13. Paulys Test on intact protein
The principle involved is the basic (left) and hydrolysate (right)
PH-BIOCHEM Expt 2 2FPH Group 1
7
CONCLUSION
The qualitative color reactions
performed on the intact protein and
hydrolysate helped predict the amino
acid component of casein. In the
Biuret test, the hydrolysate produced
a negative result. This shows that
peptide bonds were destroyed during
acid hydrolysis. In the Xanthoproteic
test, both samples produced a positive
result indicating that casein contains
an aromatic group. But in the Millons
test, both samples showed a negative
result indicating that tyrosine may not
be present in casein. In the Hopkins-
Cole test, both samples showed a
negative result indicating the absence
of tryptophan. But acid hydrolysis
completely destroys tryptophan,
presenting the possibility that
tryptophan may have been present in
casein.[1] In both Nitroprusside and
Fohls test, both samples showed a
positive result indicating that casein
contains cysteine and methionine.
Qualitative color reactions is a
means of identifying the amino acids
present in a protein. But these tests
proved to be limited and
disadvantageous compared to other
ways of characterization. Proteins
contain more than 100 amino acids
making it difficult to identify its
constituents with qualitative tests
alone. Sources of errors such as cross-
contamination of reagents, improper
isolation of protein, and the effect of
acid hydrolysis on the components
make qualitative tests insufficient in
identifying the amino acid component
of the protein.
Although the tests did not fully
predict the amino acid component of
the protein, it gave an insight as to
what amino acids may be present in
the protein.
REFERENCES
[1] Amino Acid Analysis. (n.d.).
Retrieved from Iowa State
University:
http://www.protein.iastate.edu/a
aa.html
[2] Austin Peay State University
Department of Chemistry. (n.d.).
Isolation of Casein from Milk.
Retrieved from
https://www.apsu.edu/sites/apsu
.edu/files/chemistry/SP11_1021_
ISOLATION_OF_PROTEINS_FROM
_MILK.pdf
[3] Campbell, M. K., & Farrell, S. O.
(2013). Biochemistry Eighth
Edition. Cengage Learning.
[4] Clark, J. (2004). Hydrolysing
Amides. Retrieved from
http://www.chemguide.co.uk/org
anicprops/amides/hydrolysis.ht
ml
[5] Das, D. (1978). Biochemistry.
Academic Publishers.
[6] Indian Agricultural Statistics
Research Institute. (2012,
September 19). Lesson 19:
Qualitative Test for Proteins.
Retrieved from Organic
Chemistry:
http://ecoursesonline.iasri.res.in
/mod/page/view.php?id=4188
[7] Isoelectric Precipitation of Protein:
Casein from Milk. (2011).
Retrieved from Amrita Vishwa
Vidyapeetham Virtual Lab:
http://vlab.amrita.edu/?
PH-BIOCHEM Expt 2 2FPH Group 1
8

sub=3&brch=63&sim=158&cnt proteins and what do they do?

=1 Retrieved from Genetics Home
Reference:
[8] Tiwari, A. (2013, July 18).
https://ghr.nlm.nih.gov/primer/h
Qualitative Tests for Proteins
owgeneswork/protein
and Amino Acids. Retrieved
from
http://www.hillagric.ac.in/edu/co
vas/vpb/Practical%20Manual
%20VPB%20112.pdf
[9] U.S. National Library of Medicine.
(2017, March 14). What are