QUALITATIVE ANALYSIS OF INTACT AND ACID HYDROLYSATE

CASEIN: COLOR REACTIONS

Jealeka Abesamis, Vanessa Agraviador, ​Fitz Banez​,

Natalie Regina Cu, Coleen De Guia
Group 1 2E Medical Technology Biochemistry Laboratory

ABSTRACT
Proteins play many critical roles in our body and do most of the work in cells needed for function,
structure, and regulation of the body tissues and organs. In this experiment, proteins, certain amino
acids, and amines were subjected to different qualitative color reactions tests, namely Biuret test,
Ninhydrin test, Xanthoproteic test, Millon’s test, Hopkins-Cole test, Sakaguchi test, Nitroprusside test,
Fohl’s test, Pauly’s test, and test for Amides. The intact protein tested positive for all tests, while the
acidic hydrolysate tested positive for the Ninhydrin Test, Xanthoproteic Test, Millon’s Test, Sakaguchi
Test, Nitroprusside Test, Test for Amides, and Pauly’s Test. The enzymatic hydrolysate tested positive
for the Ninhydrin Test, Nitroprusside Test, Test for Amides, and Pauly’s Test.

INTRODUCTION
Color reaction test of proteins is a j. 0.02% Naphthol solution
chemical reaction used to convert colorless k. 1% Sulfanilic acid
chemical compounds into colored derivatives l. 0.1% Ninhydrin solution
which can be observed visually. It is used to m. Conc. HNO3
determine the presence of peptide bonds and to n. Conc. H2SO4
characterize the reactivity groups of both free o. Hopkins-Cole reagent
amino acids and proteins, which are the p. Millon’s Reagent
reactions for side chains, α-amino acid, and the
α-carboxyl groups [1].
B. Procedure
The inclusion of a colored reagent causes For each test conducted, separate test
the sample to absorb and react with the colored tubes were used for intact protein solution and
product which leads to a chemical reaction hydrolyzed sample.
between two compounds. By adding two
different organic compounds, the electrons Biuret Test
between substances are rearranged which This is a chemical test used for detecting
result to a new formed substance, which causes the presence of serine and threonine. To
the change of color precipitate of an organic conduct this test, 20 drops of 2.5 M NaOH must
compound [2]. be added to the samples. Then, 2-3 drops of
0.1 M CuSO​4​ solution.
This experiment aims to show the amino
acids present in each sample, namely the intact Ninhydrin Test
protein, the enzymatic hydrolysate of casein This is a general test and thus given by
and the acid hydrolysate of casein. all amino acids. This test is due to a reaction
between a amino group of free amino acid and
ninhydrin. To conduct this test, 6-10 drops of
EXPERIMENTAL
0.1% ninhydrin solution was placed into the
A. Test Compound/s (or Sample/s) used
diluted samples. Then, the test tube was heated
​The following reagents are used for the
in a boiling water bath.
qualitative color reactions.
a. 2.5M, 3M, and 6M NaOH
Xanthoproteic Test
b. 10%, and 20% NaOH
This is a test for the presence of phenol
c. Conc. NaOH solution
group. To conduct this test, 10 drops of
d. 5% NaNO2
concentrated HNO​3 was slowly added to the
e. 10% Na2CO3
diluted samples. Then, 10 drops of concentrated
f. 2% NaOBr
NaOH was also added and mixed all together.
g. 0.1 M CuSO4
h. 5% (CH3COO)2Pb
i. 2% Nitroprusside solution
Millon’s Test appearance of the red coloration were recorded
This is a test for the presence of Phenolic if it appears.
group. To conduct this test, 5 drops of Millon’s
reagent was added to the diluted samples. RESULTS AND DISCUSSION
​The intact protein, acid hydrolysate, and
Hopkins-Cole Test enzymatic hydrolysate were subjected to
This is a test for the presence of different tests, namely Biuret Test, Ninhydrin
Aldehydes. To conduct this test, 20 drops of Test, Xanthoproteic Test, Millon’s Test,
Hopkins-Cole reagent was added to the Hopkins-Cole Test, Sakaguchi Test,
samples. Also, 20 drops of the concentrated Nitroprusside Test, Fohl’s Test, Test for Amides,
H​2​SO​4 was added along the side of the test tube and Pauly’s test yielding the following results.
while it was inclined.

Sakaguchi Test Table 1. Results of the Intact Protein Solution,

This is a test for the presence of Acid Hydrolysate, and Enzymatic Hydrolysate on
guanidinium group. To conduct this test, 10 the Biuret Test
drops of the 10% NaOH and 10 drops of the
0.02% naphthol solution was added to the Biuret Test Result
samples. It was then mixed and steadied for 3
minutes. After 3 minutes, 3 drops of the 2%
NaOBr was added and mixed, noting the color
that was produced.
Intact Protein light purple
Solution solution
Nitroprusside Test
This is a test for the presence of
Cysteine, a sulfhydryl group. To conduct this
test, 0.5 mL of the 3 M NaOH was added to 0.5
mL of the sample. Followed by 0.25% mL of the
2% nitroprusside solution which was added to
the solution. The color produced is taken note if
the solution turns red. Hydrolyzed light blue
Sample
(Acidic) solution
Fohl’s Test
This is another test for the presence of S
containing amino acid. To conduct this test, 5
drops of 30% NaOH and 2 drops of the 5% Pb
(CH3COO)2 was added to the samples. The
tube was then placed in a boiling water bath
where the appearance of dark (or black or Hydrolyzed pale blue
brown) sediments was noted. Sample
(Enzymatic) solution
Test for Amides
This is a test for the presence of
Asparagine and Glutamine. To conduct this test,
1 mL of the 20% NaOH was added to 10 drops
of the sample. The test tube was then placed in The Biuret test is a test accounting for
a boiling water bath. To test the evolution of the presence of a peptide bond. Thus, this is a
gas while heating, a moistened red litmus paper test which yield a positive result for proteins but
was placed over the mouth of the test tube and not amino acids. The formation of a cupric ion
the result was noted. accounts for the positive violet-pink complex in
the solution. Based on the results, only the
Pauly’s Test intact protein reacted with the reagent sodium
This is a test for the presence of hydroxide, indicating the presence of a peptide
Histidine and Tyrosine. To conduct this test, 5 bond. Meanwhile, the enzymatic hydrolysate
drops of the sample and 3-5 drops of the 10% and the acidic hydrolysate showed a negative
NaCO3 was added to the diazgo reagent. The result, indicating the absence of a peptide bond.
Table 2. Results of the Intact Protein Solution,
white
Acid Hydrolysate, and Enzymatic Hydrolysate on
solution
the Ninhydrin Test
with dark
Ninhydrin Result orange ppt
Test
HNO3:
bright
yellow
Hydrolyzed solution
Intact Protein blue violet Sample (Acidic)
Solution ppt NaOH:
red-orange
solution

Hydrolyzed Hydrolyzed
Sample Sample colorless
violet solution (Enzymatic)
(Acidic)

The Xanthoproteic test indicates the

presence of a phenyl ring, forming a
yellow-colored aromatic nitro compound upon
Hydrolyzed deep purple
Sample reaction with concentrated nitric acid as a
(Enzymatic) coloration positive result. Based on the results gathered
from the experiment, both the intact protein
and the acid hydrolysate tested positive for the
presence of a phenyl ring, while the enzymatic
hydrolysate tested negative.
Alpha-amino acids react with ninhydrin
to form a blue to purple colored complex. Based Table 4. Results of the Intact Protein Solution,
on the results, all the samples gave a positive Acid Hydrolysate, and Enzymatic Hydrolysate on
result, denoting the presence of alpha-amino the Millon’s Test
acids in all three solutions. Millon’s Test Result

Table 3. Results of the Intact Protein Solution,

Acid Hydrolysate, and Enzymatic Hydrolysate on
the Xanthoproteic Test
Xanthoproteic Result Intact Protein brick red
Test Solution color

HNO3:
grayish
white
solution
with
Intact Protein yellow-whit Hydrolyzed
Solution e Sample
red complex
amorphous (Acidic)
ppt

NaOH:
grayish
 same result appeared in the enzymatic
hydrolysate. This led to the testing of the
reagent against a standard. Upon doing so, the
Hydrolyzed solution did not produce a violet-colored ring as
Sample colorless well, proving that the reagent was
(Enzymatic)
contaminated; thus, accounting for the absence
of a violet-colored ring in the enzymatic
hydrolysate.

Millon’s test is based on the reaction of Table 6.​ Results of the Intact Protein Solution,
tyrosine with Millon’s reagent, mercurous Acid Hydrolysate, and Enzymatic Hydrolysate on
nitrate in nitric acid. A positive result of a flesh the Sakaguchi Test
to red complex upon heating denotes the Sakaguchi Result
presence of tyrosine in the solution. Based on Test
the results, the acidic hydrolysate and the intact
protein contain tyrosine in their solution.

Table 5. ​Results of the Intact Protein Solution,

Acid Hydrolysate, and Enzymatic Hydrolysate on Intact Protein red-orange
the Hopkins-Cole Test Solution solution
Hopkins-Cole Result
Test

Intact Protein light purple Hydrolyzed

Sample orange
Solution interphase
(Acidic) solution

Hydrolyzed colorless Hydrolyzed

Sample (Acidic) solution, no Sample colorless
violet ring (Enzymatic)

The Sakaguchi test aims to verify the

presence of arginine in a compound or solution.
Hydrolyzed colorless, no A positive result of a red-orange to red-colored
Sample violet-colore solution forms when the guanido group of
(Enzymatic) d ring arginine reacts with naphthol in the presence of
an oxidizing agent, undergoing a condensation
reaction. The sample which gave the closest to
a positive result was the intact protein, while
the acidic hydrolysate produced an
The Hopkins-Cole test is performed to orange-toned solution. Lastly, the enzymatic
determine the presence of tryptophan. Based hydrolysate did not produce a color reaction.
on the results, only the intact protein solution
contains tryptophan in its solution. However,
this is not supposed to be the case. Despite
repeating the procedure a couple of times, the
Table 7. ​Results of the Intact Protein Solution,
Acid Hydrolysate, and Enzymatic Hydrolysate on
the Nitroprusside Test
Nitroprusside Result
Test

(seen on left)
Intact Protein red-orange
Solution solution

Hydrolyzed
Sample
colorless
(seen on right) (Acidic)

Hydrolyzed red-orange
Sample (Acidic)
solution
Hydrolyzed
Sample colorless
(Enzymatic)

Hydrolyzed The Fohl’s test is performed to detect the

red-orange
Sample presence of a sulfur-containing amino acid. The
(Enzymatic) solution
degradation and substitution reaction forming
lead (II) sulfide accounts for the positive result
of a brown to black precipitate. Based on the
results, only the intact protein solution garnered
a positive result of black sediments.
The Nitroprusside test yields a positive
Table 9. ​Results of the Intact Protein Solution,
result in the form of a red solution in the
Acid Hydrolysate, and Enzymatic Hydrolysate on
presence of a thiol group in cysteine present in
the Test for Amides
the compound being tested. Although the three
samples produced a more orange-toned Test for Result
solution, the hint of red color is due to the low Amides
sulfur content in casein.
Intact Protein red litmus paper turned blue;
Table 8.​ Results of the Intact Protein Solution, Solution blue litmus paper did not
Acid Hydrolysate, and Enzymatic Hydrolysate on change in color
the Fohl’s Test
Hydrolyzed red litmus paper turned blue;
Fohl’s Test Result Sample
blue litmus paper did not
(Acidic)
dark brown change in color
Intact Protein
solution with
Solution
black ppt Hydrolyzed red litmus paper turned blue;
Sample blue litmus paper did not
(Enzymatic) change in color

The test for amides is performed to

detect the presence of a primary, secondary,
tertiary nitriles, asparagine, or glutamine. The positive for the Ninhydrin Test, Xanthoproteic
mechanism behind this test is basic hydrolysis. Test, Millon’s Test, Sakaguchi Test,
As the litmus paper is exposed to the gas Nitroprusside Test, Test for Amides, Pauly’s
evolving from the heated test tube, a positive Test. The enzymatic hydrolysate tested positive
result would change red litmus paper to blue for the Ninhydrin Test, Nitroprusside Test, Test
and leave blue litmus paper for Amides, and Pauly’s Test.
unchanged–denoting the presence of a base. As The main possible source of error for the
seen in the table of results, all three samples conducted experiment would be the use of
tested positive for the presence of an amide. unclean lab equipment such as droppers, test
tubes, etc. Performing the experiment, it was
Table 10.​ Results of the Intact Protein also noted that certain tests gave a false
Solution, Acid Hydrolysate, and Enzymatic negative result because of the contamination of
Hydrolysate on the Pauly’s Test the reagents used. This was verified through
the use of a standard, an amino acid, which
Pauly’s Test Result
tested negative for the Hopkins-Cole Test.

REFERENCES
[1] Crisostomo, A., et al
Intact Protein
red solution Laboratory Manual in General Biochemistry
Solution
Quezon City: C & E Publishing, Inc.

[2] Study. (n.d.) Retrieved March 23, 2019

from
https://study.com/academy/answer/what-does-
a-color-change-indicate-the-formation-of-in-che
mistry.html
Hydrolyzed
Sample red-orange
(Acidic) solution From books
Crisostomo, A., et al
Laboratory Manual in General Biochemistry
Quezon City: C & E Publishing, Inc.

From the internet (on-line)

Study. (n.d.) Retrieved March 23, 2019 from
https://study.com/academy/answer/what-does-
Hydrolyzed red-orange a-color-change-indicate-the-formation-of-in-che
Sample mistry.html
(Enzymatic) solution

Pauly’s test is performed to detect the

presence of tyrosine and histidine. Azo dyes are
formed when tyrosine or histidine is coupled
with diazonium salt in an alkaline condition.
Diazotization of sulfanilic acid is observed in the
presence of sodium nitrite and sodium nitrate.
This forms the positive result of a red solution
which is most evident in the intact protein. The
other two samples, the acidic hydrolysate and
the enzymatic hydrolysate, only showed a hint
of red but mostly orange solution.

CONCLUSION
The intact protein tested positive for all
tests, while the acidic hydrolysate tested