QUALITATIVE COLOR REACTION OF INTACT PROTEIN: PROTEIN AND ITS

HYDROLYSATE
Ronia Bianca G. De Leon, Jethro Kyle C. De Vera, Evan Paula F. De Villa,
Ayla Jania B. Dizon, Mark Kevin G. Flores, Bianca E. Gabagat
Group 3
2BMT
Biochemistry Laboratory

ABSTRACT
Our objective in this experiment is to isolate the following proteins: gluten from wheat flour by their difference in
solubility, casein and albumin from milk by isoelectric precipitation and heat denaturation, and myoglobin from beef
muscle by salt-induced precipitation and to examine methodically the chemical groups responsible for the color
reactions that took place and to explain the principles concerned with each test. The Biuret Test to indicate the
presence of peptide bonds, Ninhydrin Test to identify amino acids having free amino group and free carboxylic acids,
Xanthoproteic Test to detect side chains of aromatic amino acids, Millons Test to determine tyrosine and phenolic
groups, Hopkins-Cole Test to identify tryptophan residues, Sakaguchi Test to detect the presence of arginine,
Nitroprusside Test and Fohls Test to detect the presence of sulfur containing amino acids, Test for Amides to
determine R-groups of asparagine and glutamine that are present, and Pauly Test to detect imidazole ring containing
amino acids, were accomplished. Differences were detected in the results of the color reactions of the intact protein
and those of the acidic, basic and enzymatic hydrolysates.

INTRODUCTION
There are two ways to go about an analysis;
qualitative
analysis,
and
quantitative
analysis. Qualitative analysis often involves the
study of behavior and the substances found in a
certain sample. This type of analysis is more
concerned with the non-numerical characteristics
of the sample. On the other hand, quantitative
analysis is based on the numerical data which
involve the measurements and amounts of each
component obtained from a sample.
Proteins are probably the most important class
of biochemical molecules, although lipids and
carbohydrates are also essential for life. They are
the basis for the major structural components of
animal and human tissue.
Each protein within the body has a specific
function. Some proteins are involved in structural
support, while others are involved in bodily
movement, or in defense against germs. Proteins
are natural polymer molecules consisting of
amino acid units. The number of amino acids in
proteins may range from two to several
thousands. Amino acids have an array of
chemically reactive groups. The reactions for side
chains, -amino, and carboxyl groups can be
used to characterize both free amino acids and
proteins.
Intact protein of casein, albumin, gluten and
myoglobin were isolated from different sources
and samples were also hydrolyzed as a
preparation for the qualitative color reaction that
will be done through numerous tests.
The objective of this experiment is to analyze
chemical groups responsible for color reactions
and explain the principle involved in each test.

EXPERIMENTAL

A. Sample used
Intact protein and hydrolysate of: Casein,
Albumin, Gluten, and Myoglobin.

B. Procedure
Ten sample test tubes containing each of the
intact protein were prepared by adding 0.5 g of
the intact protein to 1mL of distilled water.
Another ten test tubes containing each of the
protein hydrolysate were prepared by adding 0.5
ml of the hydrolysate to 1 mL of distilled water.
1. Biuret test
20 drops of 2.5 M NaOH was added to a test
tube containing the intact protein and another 20
drops were added to the test tube containing the
enzymatic hydrolysate. Then to each test tube,
2-3 drops of 0.1 M CuSO solution were added.
Both test tubes were shaken and the color was
noted.
2. Ninhydrin Test
In each test tube, 6-10 drops of 0.1%
ninhydrin solution were added into the intact
protein and enzymatic hydrolysate. Both test
tubes were then heated in a boiling water bath.
3. Xanthoproteic Test
Ten drops of concentrated HNO3 solution was
slowly added to the diluted samples and were
mixed. Then, 10 drops of concentrated NaOH was
added and the color was noted.
4. Millons Test
To each of the diluted samples, 5 drops of
Millons reagent were added while noting the
change in color.
5. Hopkins-Cole Test
To the diluted samples, 20 drops of HopkinsCole reagent was slowly added and mixed well.
The test tubes were then inclined the test tube
and 20 drops of concentrated H2SO4 was added

along the side. The change in color was then

noted.
6. Sakaguchi Test
To each of the diluted samples, 10 drops of
10% NaOH and 10 drops of 0.02% naphthol
solution was added and the test tubes were then
left untouched for 3 minutes.
7. Nitroprusside Test
0.5 mL of 3M NaOH was added to the diluted
samples. Then, 0.25 mL of 2% of nitroprusside
solution was added.
8. Fohls Test
Five drops of 30% NaOH and 2 drops of 5%
(CH3COO)2 Pb was added to the diluted samples.
Both test tubes were then places in a boiling
water bath and the change in color was observed.
9. Test for Amides
To each of the diluted samples, 1 mL of 20%
NaOH were added and both test tubes were
placed in a boiling water bath. While in the water
bath, a red litmus paper was held at the opening
of each test tube to test for the evolution of gas.
10. Pauly Test
First, the diazo reagent was prepared by
mixing 3-5 drops of 1% sulfanilic acid with 3
drops of 5% NaNO2 solution. Then, 5 drops of the
sample and 3-5 drops of 10% Na2CO3 were added
to the diazo reagent. A red coloration was noted.

RESULTS AND DISCUSSION

In this experiment the different proteins were
isolated from their sources and its intact protein
was used in the hydrolysis experiment and
produced
acidic,
basic,
and
enzymatic
hydrolysate of casein, albumin, gluten and
myoglobin underwent different reactions to
detect the presence of specific amino acids. Since
amino acids have a variety of chemically reactive
groups, the following tests were conducted to
detect the presence of certain amino acids in
casein, albumin, gluten and myoglobin.
1. Biuret Test
The biuret test is used to detect the presence
of peptide bonds, the positive result of which will
produce a pink to violet coloration and a blue
solution for a negative indication.
As shown in Table 1, the intact protein of
casein, albumin, gluten and myoglobin all showed
the positive indication for the presence of peptide
bonds. The different reactions of the acidic
hydrolysate of casein, albumin, gluten and
myoglobin turned out to have no presence of
peptide bonds as well as the basic hydrolysate of
the proteins. For the enzymatic hydrolysate, only
casein, albumin and gluten underwent this
experiment. The end results showed that only
albumin is positive for the presence of the
peptide bonds.

It goes to show that the violet/purple color

present in the solution is due to when the cupric
ion, in a basic solution, is added to any polymer
such as proteins, which contains multiple amide
bonds.
Protein

Intact
Protein

Acidic
Hydrolysate

Basic
Hydrolysate

Enzymatic
Hydrolysate

Casein

Light
violet

Light
blue

Murky
brown

Light blue

Albumin

Light
violet

Bluegray

Murky
brown

Purple

Gluten

Clear
violet

Brown

Blue

Light blue

Myoglobin

Purple

Brown

Blue

Table 1: Results of Qualitative Color

Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin, Gluten and Myoglobin for Biuret
Test
2. Ninhydrin Test
The Ninhydrin test is a test for the presence of
amino acid. The positive result of amino acid is
detected by the yielding of a purple to blue-violet
solution. All - amino acids react with ninhydrin
(triketohydrindene hydrate), a powerful oxidizing
agent to give a purple colored product
(diketohydrin) termed Rhuemanns purple. All
primary amines and ammonia react similarly but
without the liberation of carbon dioxide. The
imino acids proline and hydroxyproline also react
with ninhydrin, but they give a yellow colored
complex instead of a purple one. Besides amino
acids, other complex structures such as peptides,
peptones and proteins also react positively when
subjected to the ninhydrin reaction. [1]
Table 2 shows that casein and myoglobin were
positive for the presence of amino acid while
albumin and gluten produced a negative result.
For the acidic hydrolysate of the samples, all
showed a negative result for the presence of
amino acid. For the basic hydrolysate of casein,
albumin and gluten, table 2.3 showed that there
is a negative presence of amino acid because of
the yellow and colorless solution as their result.
The yellow solution is due to the imino acids that
react with ninhydrin. On the other hand,
myoglobin produced a violet solution indicating
the presence of amino acid in its structure. The
enzymatic hydrolysate of casein and albumin
showed the presence of amino acid while

gluten showed no presence of amino acid by

yielding a colorless solution.
Fig.1 Formation of Rheumanns Purple

Hydrolysate
Protein
Casein

Intact
Protein
Blue
violet

Acidic
Colorless

Basic
Yellow

the aromatic amino acids. So for the intact

protein and basic hydrolysate, all samples
indicated a positive result for the test. The acidic
hydrolysate of casein and albumin tested positive
for the presence of aromatic side chains while
gluten and myoglobin resulted negative. For the
enzymatic hydrolysate samples, only casein was
positive for the test among the three.
The explanation behind this is that some amino
acids
contain
aromatic
groups
that
are
derivatives of benzene such as tyrosine and
tryptophan. These aromatic groups can undergo
reactions that are characteristics of benzene and

Enzymatic
Purple

Hydrolysate
Protein

Intact
Protein

White

Yellowish
brown
Light
yellow
Dark
brown
Light
yellow
Brown

Yellow

Brown

Light
yellow
w/ppt

Light
brown

White

Albumin

Colorless

Brown

Yellow

Blueviolet

Gluten

Colorless

Brown

Colorless

Colorless

Myoglobin

Dark
Purple

Dark
Brown

Casein
Yellow

Albumin

Light
yellow
Yellow

Violet

Gluten

Myoglobin

Figure 1 shows the removal of the amino (NH2)

group from an amino acid to form a blue violet
complex or the Rheumanns purple.
Table 2: Results of Qualitative Color
Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin,
Gluten
and
Myoglobin
for
Ninhydrin Test
3. Xanthoproteic Test
Xanthoproteic test detects side chains of
aromatic amino acids, such as Phenylalanine,
Tyrosine and Tryptophan, respond to this test. In
the presence of concentrated nitric acid, the
aromatic phenyl ring is nitrated to give yellow
colored nitro-derivatives. At alkaline pH, the color
changes to orange due to the ionization of the
phenolic group. (see fig. 2)
In table 3, upon addition of HNO3 the results
were put above the highlighted ones which were
the end results after adding NaOH to the samples
and the basis for the indication of the presence of

Acidic

Basic

Yellow

Yellow

Yellow
Yellow

Enzymatic
Light
yellow

Colorless
Colorless

Pale
yellow

benzene derivatives. One such reaction is the

nitration of a benzene ring with nitric acid. The
amino acids that have activated benzene ring can
readily undergo nitration. This nitration reaction,
in the presence of activated benzene ring, forms
yellow product.
Fig.2 Ionization of the Phenolic group

Table 3: Results of Qualitative Color

Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin,
Gluten
and
Myoglobin
for
Xanthoproteic Test
4. Millons Test
Millons test is given by any compound
containing
a
phenolic
hydroxy
group.
Consequently, any protein containing tyrosine
andits derivatives will give a positive test of a
pink to dark-red color.
Based on the results tabulated (see table 4), all
the intact proteins responded negative to the

Hydrolysate
Hydrolysate
Protein
Casein

Albumin

Gluten

Myoglobin

Intact
Protein

Acidic

Colorless

Colorless

Yellow

Colorless

Light
yellow

Yellow

Colorless

Brown

Colorless

Colorless

Clear
yellow
w/
black
ppt

Basic

Colorless

Enzymatic
Colorless
Colorless

Protein

Intact
Protein

Acidic

Basic

Enzymatic

Casein

Purple

Colorless

Colorless

Colorless

Albumin

Colorless

Clear
brown

Light
yellow

Colorless

Gluten

Violet
ring

Brown

Peach

Colorless

Myoglobin

Clear
light
brown

Yellow
w/
dark
brown
layer

purple

Colorless

reaction as well as the acidic, basic and

enzymatic hydrolysate of casein, albumin, gluten
and myoglobin.
Table 4: Results of Qualitative Color
Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin, Gluten and Myoglobin for Millons
Test
5. Hopkins-cole Test
This test is specific for detecting tryptophan.
Violet to blue color develops when a mixture of
protein and an aldehyde is layered over conc.
sulphuric acid. A number of tests based on this
principle have been suggested; all depends on
the presence of the indoly group of tryptophan
which reacts as follows (using glyoxylic acid as an
example of an aldehyde).[2]
The results, in table 5, showed that the intact
protein of casein and gluten were positive for the
presence of tryptophan. The violet ring that was
present in the intact protein of gluten is due to
the presence of the indole group. For the acidic
and enzymatic hydrolysate results indicated the
absence of tryptophan in their composition. For
the basic hydrolysate samples, only myoglobin
was positive for the test.
Fig. 3 Formation of the violet ring

The figure above shows that the presence of

the indole ring of the tryptophan causes the
violet ring/solution in the reaction.

Table 5: Results of Qualitative Color

Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin, Gluten and Myoglobin for Hopkinscole Test
6. Sakaguchi Test
Sakaguchi test is often used for determining
the presence of arginine and other guanidyl
derivatives (glycocyamine, methylgyanidine etc.)
react with hypobromite and alpha napthol to give
a red colored product.
The results gathered (see table 6) showed that
none of the intact protein were positive for the
presence of arginine or other guanidyl derivatives
as well as the acidic and enzymatic hydrolysate.
However, for the basic hydrolysate of myoglobin,
the result was a red solution showing that
arginine or other guanidyl derivatives were
present in its composition. The red coloration is
due to the guanidine group reacting with alpha
napthol and sodium hypobromite.
Table 6: Results of Qualitative Color
Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin,
Gluten
and
Myoglobin
for
Sakaguchi Test

Hydrolysate
Protein

Intact
Protein

Acidic

Basic

Enzymatic

Casein

Yellow

Dark
Yellow

Red

Dark
Yellow

Albumin

Dark
Yellow

Yellow

Clear
Yellow

Dark
Yellow

Gluten

Slightly
turbid
yellow

Reddishbrown

Myoglobin

Light
yellow

Brown

7. Nitroprusside Test
This test is used to detecting the presence of
free thiol groups of cysteine in proteins. Proteins
with the free thiol group give a red colour when
added to sodium nitroprusside with ammonium
hydroxide.
The results were tabulated in table 7 and show
that for the intact protein, none of the samples
were positive for the presence of free thiol
groups. The acidic hydrolysate of gluten showed
a partial presence of cysteine due to the reddishbrown solution produced. For the basic
hydrolysate of casein and myoglobin, the results
were positive of the presence of free thiol groups
due to the red solution it produced however, for
the results given by the enzymatic hydrolysate of
casein, albumin and gluten, there is an absence
of cysteine in their composition.
Table

Yellow

Red

7:

Results

of

Yellow

Qualitative

Color

Hydrolysate
Protein

Intact
Protein

Acidic

Basic

Enzymatic

Casein

Colorless

Light
yellow

Light
yellow

Colorles
s

Albumin

Dark
yellow

Light
orang
e

Light
yellow

Light
yellow

Gluten

Colorless
turbid

Brown

Colorles
s

Colorles
s

Myoglobi
n

Colorless
w/ light
brown
sediments

Brown
w/
black
ppt

Red

Reaction for Intact protein and Hydrolysate

(Acidic, Basic and Enzymatic) of Casein,
Albumin,
Gluten
and
Myoglobin
for
Nitroprusside Test
8. Fohls Test
Fohls test is performed for the determination
of S- containing amino acids. The soutions were
heated for the formation of sulfide. The test
yielded a positive result, if the solution showed a
red solution or a further reaction of Na2S will lead
to a dark brown precipitate.

Fig.3 Fohls reaction

The results of the reaction with the intact
protein (see table 8) showed the presence of S-

Hydrolysate
Protein

Intact
Protein

Acidic

Basic

Casein

Red to blue
litmus
paper;
yellow

Red to
blue
litmus
paper

Blue
to red
litmus
paper

Albumin

Red to blue
litmus
paper;
brown
suspension

Red to
blue
litmus
paper

Blue
to red
litmus
paper

Red to blue
litmus
paper;
yellow

Red to
blue
litmus
paper

Red to
blue
litmus
paper

Myoglobin

Red to blue
litmus
paper

Red to
blue
litmus
paper

Red to
blue
litmus
paper

Protein

Intact
Protei
n

Acidic

Basic

Enzymatic

Casein

Red/
orange

Dark
orange

Brown

Red

Albumin

Red/
orange

Orange

Colorless

Redorange

Gluten

Redorange

Red

Red

Yelloworange

Myoglobin

Red

Red

Red

Gluten

Enzymatic
Red to
blue
litmus
paper;
colorless
Red to
blue
litmus
paper;
light
yellow
Red to
blue
litmus
paper;
colorless

The results in table 9 showed that all the intact

protein of the samples as well as their acidic
hydrolysate tested positive for the presence of a
basic component. However for the basic
hydrolysate of casein and albumin, the litmus
paper turned from blue to red, indicating a
negative result for the test while the basic
Hydrolysate
Protein

Intact
Protein

Acidic

Basic

Enzymatic

Casein

Brownish
black

Dark
brown
sediments

Brown

Light
yellow

Albumin

Brown

Light
orange

Brown

Light
brown

Gluten

Brownish
black

Blackbrown

Colorless

Colorless

Myoglobin

Brown
sediments

Brownish
orange

Colorless

Hydrolysate

containing amino acids as well as the acidic

hydrolysate. For the basic hydrolysate of the
proteins, only casein and albumin were found
positive of the presence of s- containing amino
acids while the enzymatic hydrolysate of albumin
solely showed the positive result among the
three.
The brown/black sediments were due to the
further reaction of Na2S.
Table 8: Results of Qualitative Color
Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin, Gluten and Myoglobin for Fohls
Test
9. Test for Amides
The test for amides is done for the presence of
Asparagine and Glutamine. When the red limus
paper turned blue, it indicates a basic component
is present in the protein.

hydrolysate of gluten and myoglobin and also the

enzymatic hydrolysate of the samples indicated a
positive result.
Table 9: Results of Qualitative Color
Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin, Gluten and Myoglobin for the
Amide Test
10. Pauly Test
This test is specific for the detection of
imidazole ring. The sulfanilic acid gets diazotized
in the presence of sodium nitrite and sodium
carbonate. Positive result will show yellow to dark
orange solution and residues. The residues are
histidine and / or tyrosine residues.
As shown in table 10, the intact protein and
acidic hydrolysate of the samples yielded a
positive result while the basic hydrolysate of
casein and albumin yielded a negative result. The
basic hydrolysate of gluten and myoglobin as well
as the enzymatic hydrolysate of the samples
produced a positive indication of the presence of
imidazole ring.
Table 10: Results of Qualitative Color
Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin, Gluten and Myoglobin for the Pauly
Test

REFERENCES:

[1] L. Castillo, Color Reactions of Proteins.

https://quizlet.com/8801729/color-reactions-ofproteins-flash-cards/ 3/26/2016
[2] S.P. Singh, Qualitative Analysis of Amino
Acid.

http://vlab.amrita.edu/?
sub=3&brch=63&sim=1094&cnt=6 3/26/2016