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, Millon’s 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 Fohl’s 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 A. Sample used

There are two ways to go about an analysis; Intact protein and hydrolysate of: Casein,
qualitative analysis, and quantitative Albumin, Gluten, and Myoglobin.
analysis. Qualitative analysis often involves the
study of behavior and the substances found in a B. Procedure
certain sample. This type of analysis is more Ten sample test tubes containing each of the
concerned with the non-numerical characteristics intact protein were prepared by adding 0.5 g of
of the sample. On the other hand, quantitative the intact protein to 1mL of distilled water.
analysis is based on the numerical data which Another ten test tubes containing each of the
involve the measurements and amounts of each protein hydrolysate were prepared by adding 0.5
component obtained from a sample. ml of the hydrolysate to 1 mL of distilled water.
Proteins are probably the most important class 1. Biuret test
of biochemical molecules, although lipids and 20 drops of 2.5 M NaOH was added to a test
carbohydrates are also essential for life. They are tube containing the intact protein and another 20
the basis for the major structural components of drops were added to the test tube containing the
animal and human tissue. enzymatic hydrolysate. Then to each test tube,
Each protein within the body has a specific 2-3 drops of 0.1 M CuSO solution were added.
function. Some proteins are involved in structural Both test tubes were shaken and the color was
support, while others are involved in bodily noted.
movement, or in defense against germs. Proteins 2. Ninhydrin Test
are natural polymer molecules consisting of In each test tube, 6-10 drops of 0.1%
amino acid units. The number of amino acids in ninhydrin solution were added into the intact
proteins may range from two to several protein and enzymatic hydrolysate. Both test
thousands. Amino acids have an array of tubes were then heated in a boiling water bath.
chemically reactive groups. The reactions for side 3. Xanthoproteic Test
chains, α-amino, and α–carboxyl groups can be Ten drops of concentrated HNO3 solution was
used to characterize both free amino acids and slowly added to the diluted samples and were
proteins. mixed. Then, 10 drops of concentrated NaOH was
Intact protein of casein, albumin, gluten and added and the color was noted.
myoglobin were isolated from different sources 4. Millon’s Test
and samples were also hydrolyzed as a To each of the diluted samples, 5 drops of
preparation for the qualitative color reaction that Millon’s reagent were added while noting the
will be done through numerous tests. change in color.
The objective of this experiment is to analyze 5. Hopkins-Cole Test
chemical groups responsible for color reactions To the diluted samples, 20 drops of Hopkins-
and explain the principle involved in each test. Cole reagent was slowly added and mixed well.
The test tubes were then inclined the test tube
EXPERIMENTAL and 20 drops of concentrated H2SO4 was added
along the side. The change in color was then It goes to show that the violet/purple color
noted. present in the solution is due to when the cupric
6. Sakaguchi Test ion, in a basic solution, is added to any polymer
To each of the diluted samples, 10 drops of such as proteins, which contains multiple amide
10% NaOH and 10 drops of 0.02% naphthol bonds.
solution was added and the test tubes were then Acidic Basic Enzymatic
left untouched for 3 minutes. Intact
Protein Hydro- Hydro- Hydro-
7. Nitroprusside Test Protein
lysate lysate lysate
0.5 mL of 3M NaOH was added to the diluted
samples. Then, 0.25 mL of 2% of nitroprusside Light Light Murky
Casein Light blue
solution was added. violet blue brown
8. Fohl’s Test
Five drops of 30% NaOH and 2 drops of 5% Light Blue- Murky
Albumin Purple
(CH3COO)2 Pb was added to the diluted samples. violet gray brown
Both test tubes were then places in a boiling
water bath and the change in color was observed. Clear
Gluten Brown Blue Light blue
9. Test for Amides violet
To each of the diluted samples, 1 mL of 20%
NaOH were added and both test tubes were Myoglobin Purple Brown Blue -
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. Table 1: Results of Qualitative Color
10. Pauly Test Reaction for Intact protein and Hydrolysate
First, the diazo reagent was prepared by (Acidic, Basic and Enzymatic) of Casein,
mixing 3-5 drops of 1% sulfanilic acid with 3 Albumin, Gluten and Myoglobin for Biuret
drops of 5% NaNO2 solution. Then, 5 drops of the Test
sample and 3-5 drops of 10% Na2CO3 were added 2. Ninhydrin Test
to the diazo reagent. A red coloration was noted. The Ninhydrin test is a test for the presence of
 amino acid. The positive result of amino acid is
RESULTS AND DISCUSSION detected by the yielding of a purple to blue-violet
In this experiment the different proteins were solution. All α- amino acids react with ninhydrin
isolated from their sources and its intact protein (triketohydrindene hydrate), a powerful oxidizing
was used in the hydrolysis experiment and agent to give a purple colored product
produced acidic, basic, and enzymatic (diketohydrin) termed Rhuemann’s purple. All
hydrolysate of casein, albumin, gluten and primary amines and ammonia react similarly but
myoglobin underwent different reactions to without the liberation of carbon dioxide. The
detect the presence of specific amino acids. Since imino acids proline and hydroxyproline also react
amino acids have a variety of chemically reactive with ninhydrin, but they give a yellow colored
groups, the following tests were conducted to complex instead of a purple one. Besides amino
detect the presence of certain amino acids in acids, other complex structures such as peptides,
casein, albumin, gluten and myoglobin. peptones and proteins also react positively when
1. Biuret Test subjected to the ninhydrin reaction. [1]
The biuret test is used to detect the presence Table 2 shows that casein and myoglobin were
of peptide bonds, the positive result of which will positive for the presence of  amino acid while
produce a pink to violet coloration and a blue
albumin and gluten produced a negative result.
solution for a negative indication.
For the acidic hydrolysate of the samples, all
As shown in Table 1, the intact protein of
showed a negative result for the presence of 
casein, albumin, gluten and myoglobin all showed
the positive indication for the presence of peptide amino acid. For the basic hydrolysate of casein,
bonds. The different reactions of the acidic albumin and gluten, table 2.3 showed that there
hydrolysate of casein, albumin, gluten and is a negative presence of  amino acid because of
myoglobin turned out to have no presence of the yellow and colorless solution as their result.
peptide bonds as well as the basic hydrolysate of The yellow solution is due to the imino acids that
the proteins. For the enzymatic hydrolysate, only react with ninhydrin. On the other hand,
casein, albumin and gluten underwent this myoglobin produced a violet solution indicating
experiment. The end results showed that only the presence of  amino acid in its structure. The
albumin is positive for the presence of the enzymatic hydrolysate of casein and albumin
peptide bonds. showed the presence of  amino acid while
gluten showed no presence of  amino acid by the aromatic amino acids. So for the intact
yielding a colorless solution. protein and basic hydrolysate, all samples
indicated a positive result for the test. The acidic
Fig.1 Formation of Rheumann’s Purple 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
Hydrolysate reactions that are characteristics of benzene and
Intact Enzy-
Protein Acidic Basic Hydrolysate
Protein matic
Intact Enzy-
Blue Protein Acidic Basic
Casein Colorless Yellow Purple Protein matic
violet Yellowish
White Light
brown
Blue- Casein Yellow yellow
Albumin Colorless Brown Yellow Light
violet Yellow
yellow
Light Dark
Color- Color-
Gluten Colorless Brown Colorless yellow brown Yellow
less Albumin less
Light
Yellow
Dark Dark yellow
Myoglobin Violet - White Brown Yellow Color-
Purple Brown
Gluten less
Yellow Brown Yellow

Light Pale
Light
Myoglobin yellow yellow
brown -
w/ppt
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
Figure 1 shows the removal of the  amino (NH2) yellow product.
group from an amino acid to form a blue violet
complex or the Rheumann’s purple. Fig.2 Ionization of the Phenolic group

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 Table 3: Results of Qualitative Color
3. Xanthoproteic Test Reaction for Intact protein and Hydrolysate
Xanthoproteic test detects side chains of (Acidic, Basic and Enzymatic) of Casein,
aromatic amino acids, such as Phenylalanine, Albumin, Gluten and Myoglobin for
Tyrosine and Tryptophan, respond to this test. In Xanthoproteic Test
the presence of concentrated nitric acid, the
aromatic phenyl ring is nitrated to give yellow 4. Millon’s Test
colored nitro-derivatives. At alkaline pH, the color Millon’s test is given by any compound
changes to orange due to the ionization of the containing a phenolic hydroxy group.
phenolic group. (see fig. 2) Consequently, any protein containing tyrosine
In table 3, upon addition of HNO3 the results andits derivatives will give a positive test of a
were put above the highlighted ones which were pink to dark-red color.
the end results after adding NaOH to the samples Based on the results tabulated (see table 4), all
and the basis for the indication of the presence of the intact proteins responded negative to the
 Hydrolysate
Intact Enzy-
Hydrolysate Protein Acidic Basic
Protein matic
Intact Enzy-
Protein Acidic Basic
Protein matic Color- Color-
Casein Purple Colorless
less less
Color- Color- Color-
Casein Yellow
less less less Color- Clear Light Color-
Albumin
less brown yellow less
Color- Light Color-
Albumin Yellow
less yellow less Violet Color-
Gluten Brown Peach
ring less
Color- Color- Color-
Gluten Brown Yellow
less less less
Clear w/
Clear
Myoglobin light dark purple -
yellow
Color- Color- brown brown
Myoglobin w/ -
less less layer
black
ppt
reaction as well as the acidic, basic and Table 5: Results of Qualitative Color
enzymatic hydrolysate of casein, albumin, gluten Reaction for Intact protein and Hydrolysate
and myoglobin. (Acidic, Basic and Enzymatic) of Casein,
Albumin, Gluten and Myoglobin for Hopkins-
Table 4: Results of Qualitative Color cole Test
Reaction for Intact protein and Hydrolysate 6. Sakaguchi Test
(Acidic, Basic and Enzymatic) of Casein, Sakaguchi test is often used for determining
Albumin, Gluten and Myoglobin for Millon’s the presence of arginine and other guanidyl
Test derivatives (glycocyamine, methylgyanidine etc.)
5. Hopkins-cole Test react with hypobromite and alpha napthol to give
This test is specific for detecting tryptophan. a red colored product.
Violet to blue color develops when a mixture of The results gathered (see table 6) showed that
protein and an aldehyde is layered over conc. none of the intact protein were positive for the
sulphuric acid. A number of tests based on this presence of arginine or other guanidyl derivatives
principle have been suggested; all depends on as well as the acidic and enzymatic hydrolysate.
the presence of the indoly group of tryptophan However, for the basic hydrolysate of myoglobin,
which reacts as follows (using glyoxylic acid as an the result was a red solution showing that
example of an aldehyde).[2] arginine or other guanidyl derivatives were
The results, in table 5, showed that the intact present in its composition. The red coloration is
protein of casein and gluten were positive for the due to the guanidine group reacting with alpha
presence of tryptophan. The violet ring that was napthol and sodium hypobromite.
present in the intact protein of gluten is due to
the presence of the indole group. For the acidic Table 6: Results of Qualitative Color
and enzymatic hydrolysate results indicated the Reaction for Intact protein and Hydrolysate
absence of tryptophan in their composition. For (Acidic, Basic and Enzymatic) of Casein,
the basic hydrolysate samples, only myoglobin Albumin, Gluten and Myoglobin for
was positive for the test. Sakaguchi 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.
 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
Hydrolysate a partial presence of cysteine due to the reddish-
Intact Enzy- brown solution produced. For the basic
Protein Acidic Basic hydrolysate of casein and myoglobin, the results
Protein matic
were positive of the presence of free thiol groups
Dark Dark due to the red solution it produced however, for
Casein Yellow Red
Yellow Yellow the results given by the enzymatic hydrolysate of
casein, albumin and gluten, there is an absence
Dark Clear Dark of cysteine in their composition.
Albumin Yellow
Yellow Yellow Yellow

Slightly Table 7: Results of Qualitative Color

Reddish-
Gluten turbid Yellow Yellow
brown Hydrolysate
yellow
Intact Enzy-
Protein Acidic Basic
Light Protein matic
Myoglobin Brown Red -
yellow
Light Light Colorles
Casein Colorless
yellow yellow s
Light
Dark Light Light
Albumin orang
yellow yellow yellow
e
Colorless Colorles Colorles
Gluten Brown
turbid s s
Colorless Brown
Myoglobi w/ light w/
Red -
n brown black
sediments ppt
Reaction for Intact protein and Hydrolysate
(Acidic, Basic and Enzymatic) of Casein,
Albumin, Gluten and Myoglobin for
Nitroprusside Test
8. Fohl’s Test
Fohl’s 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 Fohl’s reaction

The results of the reaction with the intact
protein (see table 8) showed the presence of S-
 Hydrolysate
Intact Enzy- The results in table 9 showed that all the intact
Protein Acidic Basic protein of the samples as well as their acidic
Protein matic
Red to hydrolysate tested positive for the presence of a
Red to blue Red to Blue basic component. However for the basic
blue
litmus blue to red hydrolysate of casein and albumin, the litmus
Casein litmus
paper; litmus litmus paper turned from blue to red, indicating a
paper;
yellow paper paper negative result for the test while the basic
colorless
Red to Hydrolysate
Red to blue Intact Enzy-
Red to Blue blue Protein Acidic Basic
litmus Protein matic
blue to red litmus
Albumin paper;
litmus litmus paper; Dark
brown
paper paper light Brownish brown Light
suspension Casein Brown
yellow black sedi- yellow
Red to ments
Red to blue Red to Red to
blue
litmus blue blue Light Light
Gluten litmus Albumin Brown Brown
paper; litmus litmus orange brown
paper;
yellow paper paper
colorless Brownish Black- Color- Color-
Red to Red to Gluten
Red to blue black brown less less
Myoglo- blue blue
litmus -
bin litmus litmus Brown Brownish Color-
paper Myoglobin -
paper paper sediments orange less

Hydrolysate hydrolysate of gluten and myoglobin and also the

Intact enzymatic hydrolysate of the samples indicated a
Enzy-
Protein Protei Acidic Basic positive result.
matic
n
Red/ Dark
Table 9: Results of Qualitative Color
Casein Brown Red Reaction for Intact protein and Hydrolysate
orange orange
(Acidic, Basic and Enzymatic) of Casein,
Red/ Color- Red- Albumin, Gluten and Myoglobin for the
Albumin Orange Amide Test
orange less orange

Red- Yellow- 10. Pauly Test

Gluten Red Red
orange orange This test is specific for the detection of
Myoglo- imidazole ring. The sulfanilic acid gets diazotized
Red Red Red - in the presence of sodium nitrite and sodium
bin
carbonate. Positive result will show yellow to dark
containing amino acids as well as the acidic orange solution and residues. The residues are
hydrolysate. For the basic hydrolysate of the histidine and / or tyrosine residues.
proteins, only casein and albumin were found As shown in table 10, the intact protein and
positive of the presence of s- containing amino acidic hydrolysate of the samples yielded a
acids while the enzymatic hydrolysate of albumin positive result while the basic hydrolysate of
solely showed the positive result among the casein and albumin yielded a negative result. The
three. basic hydrolysate of gluten and myoglobin as well
The brown/black sediments were due to the as the enzymatic hydrolysate of the samples
further reaction of Na2S. produced a positive indication of the presence of
imidazole ring.
Table 8: Results of Qualitative Color
Reaction for Intact protein and Hydrolysate Table 10: Results of Qualitative Color
(Acidic, Basic and Enzymatic) of Casein, Reaction for Intact protein and Hydrolysate
Albumin, Gluten and Myoglobin for Fohl’s (Acidic, Basic and Enzymatic) of Casein,
Test Albumin, Gluten and Myoglobin for the Pauly
9. Test for Amides Test
The test for amides is done for the presence of
REFERENCES:
Asparagine and Glutamine. When the red limus
paper turned blue, it indicates a basic component
[1] L. Castillo, Color Reactions of Proteins.
is present in the protein.
https://quizlet.com/8801729/color-reactions-of- http://vlab.amrita.edu/?
proteins-flash-cards/ 3/26/2016 sub=3&brch=63&sim=1094&cnt=6 3/26/2016

[2] S.P. Singh, Qualitative Analysis of Amino

Acid.