QUALITATIVE COLOR REACTIONS OF INTACT PROTEIN AND ITS HYDROLYSATES

(ALBUMIN)
Zoe Angela M. Espinosa, Ma. Pamela Rica M. Fajardo, Emmanuel Joshua M. Garcia,
Liana Celina D. Geluz, Jillian Pamela D. Go, and Jennifer Mae C. Heredia
Group 4
2A- Medical Technology
CHEM 600 Laboratory

ABSTRACT
The qualitative color reactions of albumin were tested through color reactions confirming the presence of certain
amino acids in both the intact albumin and in its hydrolysates. The Biuret test confirmed the presence of protein in all
samples with each giving a blue-violet coloration, while the Ninhydrin test detected the presence of amino acids
completely hydrolyzed in the basic hydrolysate, as it was the only sample which gave a blue-violet solution. The
Xanthoproteic test detected the presence of an aromatic amino (tyrosine or tryptophan) as positive results (yellow
solution) were observed in albumin. This aromatic amino acid was specified through the Millon test and through the
Pauly test as all samples tested negative for both reactions specific for tyrosine. The presence of tryptophan was then
confirmed through the Hopkins-Cole test which gave a red ring at the junction instead of a purple ring. In taking note
of the errors committed in the experiment, this was already considered a positive result. The Sakaguchi test and the
the test for amide both confirmed the presence of arginine as a non-terminal amino acid, in giving a negative result
for the intact protein but a positive result for at least one of the hydrolysates. The Fohls test detected the presence of
sulfur-containing samples (either cysteine or methionine) as a dark brown solution was observed from the results.
This amino acid was further specified as methionine with the negative result of the basic hydrolysate in Fohls test and
with the negative results of the Nitroprusside test specific for cysteine. The color test reactions were able to detect the
presence of tryptophan, arginine, and methionine in albumin.

INTRODUCTION
Albumin is a water-soluble, heat coagulable
protein. Albumin is present in egg white (egg
albumin), milk (lactalbumin) and in serum. Egg
albumin and lactalbumin are glycoconjugated
proteins, whereas serum albumin is a simple
protein. Albumin is a protein of high biological
value with all essential amino acids [10].
Amino acids have a variety of chemically
reactive groups. The reactions for side chains, amino and -carboxyl groups can be used to
characterize both free amino acids and proteins
[2]. In characterizing the amino acids present in
albumin, the principle behind qualitative color
reactions is employed through different tests and
reagents.
Biuret test is used to detect the presence of a
protein or peptides. Its positive result is a violet
coloration which means that it is a substance
(protein) that contains not less than two peptide
linkages [9].
Ninhydrin test is a typical test for an -amino
acid. Its positive result is a blue to blue-violet to
violet coloration with -amino acids or yellow
coloration with cyclic amino acids such as proline
and hydroxyproline [1].
Xanthroproteic test detects side chains of
aromatic amino acids. Its positive result is a
yellow coloration on heating with acid forming
nitro derivatives or an orange coloration when
made alkaline with the base forming sodium salts
of these derivatives [5].
Millons test is a color reaction test
using Millon's
reagent to
detect
phenolic

compounds such as tyrosine. Its positive result is

a red precipitate or a red solution [5].
Hopkins-Cole test is a specific for tryptophan.
Its positive result is a pink to purple interface [9].
Sakaguchi test is a specific test for arginine. Its
positive result is an orange to red solution [1].
Nitroprusside test is a specific test for cysteine.
Its positive result is a red solution [9].
Fohls test is used to detect sulfur-containing
proteins. Its positive result is a brown-black
precipitate.
The test for amides is used to detect R-groups
of asparagine and glutamine [5]. Its positive
result is a red to blue litmus paper.
Pauly test is specific for the detection of
Tryptophan or Histidine [6]. Its positive result is a
red or purple coloration.
The objectives of the experiment are 1) to
analzye chemical groups responsible for color
reactions and 2) to explain the principle involved
in each color reaction test [2].

EXPERIMENTAL
A. Compounds Tested
Intact albumin
Hydrolyzed albumin
- Acidic hydrolysate
- Basic hydrolysate
- Enzymatic hydrolysate
B. Procedure
For each test, the sample was prepared in
separate test tubes for the intact protein solution
and for the hydrolyzed samples. The intact
protein solution was prepared by having 0.5
grams of the protein in 1 mL of distilled H2O while

0.5 mL each of the hydrolyzed samples (acid,

basic, enzymatic) was used.
1. Biuret Test
Twenty drops of 2.5 M NaOH was introduced
to the samples and mixed well. Then, 2-3 drops
of 0.1 M CuSO4 solution was added to the
solutions. The test tubes were then shaken to
mix the solution well, and the colors were noted
down.
2. Ninhydrin Test
Six to ten drops of 0.1% ninhydrin solution
was placed into diluted samples. The tube was
then heated in a boiling water bath; a blue-violet
coloration was noted down.
3. Xanthoproteic Test
Ten drops of concentrated HNO 3 was slowly
added to the diluted samples. It was mixed, and
the color of the solution was noted. Ten drops of
concentrated NaOH was slowly added. It was also
mixed, and its color was noted.
4. Millons Test
Five drops of Millons reagent was added to
the diluted samples. The change in color was
noted.
5. Hopkins-Cole Test
Twenty drops of Hopkins-Cole reagent was
slowly added to the samples. It was then mixed
well. The test tube was inclined, and along its
side, twenty drops of concentrated H2SO4 wass
slowly added. The color at its interface was
noted.
6. Sakaguchi Test
Ten drops of 10% NaOH was added. Ten drop
of 0.2% naphthol solution was also added to the
samples. It was then mixed and let stand for
three minutes. Three drops of 2% NaOBr was
added. It was then mixed, and the color
produced was noted.
7. Nitroprusside Test
A half milliliter of 3 M NaOH was added to the
0.5 mL of the sample. A quarter millliliter of 2%
nitroprusside soluytion was then added. The
formation of a red solution was noted.
8. Fohls Test
Five drops of 30% NaOH was added; two
drops of 5% (CH3COO)2Pb was then added to the
samples. The tube was placed in a boiling water
bath. After that, the appearance of dark (black or
brown) sediment was noted.
9. Test for Amides
One milliliter of 20% NaOH was added to ten
drops of the sample. The tube was placed in a
boiling water bath. The evolution of gas during
heating was tested by placing a moistened red
litmus paper over the mouth of the tube.
10.Pauly Test
The diazo reagent was prepared by mixing 35 drops of 1% sulfanilic acid with 3 drops of 5%
NaNO2 solution. Five drops of the sample and 3-5

drops of 10% Na2CO3 was added to the diazo

reagent. The appearance of a red coloration was
noted.

RESULTS AND DISCUSSION

Proteins, due to the presence of peptide bonds
and different amino acid residues, react with a
variety of reagents to form colored products.
These tests are known as color reactions of
proteins. Several of these reactions are important
in
qualitative
detection
and
quantitative
estimation of proteins and amino acids [10].
The samples used for the qualitative color
reactions include the intact protein, acidic
hydrolysate, basic hydrolysate, and enzymatic
hydrolysate of the isolated protein albumin found
in skimmed milk. The samples were all tested to
characterize and determine the functional groups
that they contain, depending on their reactions to
the reagents of each test.
Biuret test is a general test for proteins and is
a test for detecting peptide linkage. The biuret
reagent (copper sulfate in a strong base) reacts
with peptide bonds in proteins to form a blue to
violet complex known as the Biuret complex. At
least two peptide bonds are required for the
formation of this complex [1].

Figure 1. Protein-copper(II) ion complex, also

called the biuret complex
Based on the results obtained, the intact
protein showed a positive result (purple solution)
for this test since its peptide linkage is not yet
broken. This goes the same for the acidic and
enzymatic hydrolyzed samples which also
presented a positive result (purple solution) for
the biuret test. This means that the acidic and
enzymatic hydrolysis werent able to completely
hydrolyze the sample unlike in the basic
hydrolysis which presented a clear blue solution
depicting a lower intensity of color correlating to
the presence of hydrolyzed protein in the form of
amino acids.
Table 1. Results of the Biuret Test
Biuret Test

Protein
Hydrolysate

Intact Protein
Acidic
Basic
Enzymatic

Purple solution
Purple solution
Clear light blue solution
Purple solution

Ninhydrin is a test for detecting free -amino

groups. The covalently bonded peptide bonds
present in proteins are disrupted as they are
hydrolyzed. After completion of hydrolysis, the
hydrolyzed protein or hydrolysate protein is what
remains, containing a mixture of amino acids that
react with ninhydrin. Ninhydrin reacts with amino acids (-NH2) in proteins to give a purple
complex as illustrated in the reaction that follows,
with the exception of proline and hydroxy proline
which both give a yellow color due to the absence
of free -amino acid. Ninhydrin is most
commonly used as a forensic chemical to detect
fingerprints as amines left over from proteins,
sloughed off in fingerprints, react with ninhydrin
to give a characteristic purple color [1].

Figure 2. Ninhydrin reaction forming a purple

complex
The principle behind this test is oxidative
deamination with a free amino group, -NH 2. The
amino group is released as ammonia (NH 3). A
positive indication of this test would be a blueviolet coloration in the solution, which deepens as
the number of amino group increases. In the
results obtained, only the basic hydrolysate
tested positive for this test as the intact protein,
acidic hydrolysate, and enzymatic hydrolysate all
presented a colorless solution in the presence of
ninhydrin. It can be said that the basic
hydrolysate is the only sample which produced
the most free -amino groups as a result of its
complete hydrolysis.
Table 2. Results of the Ninhydrin Test

Figure 3. Aromatic amino acid reaction with

concentrated HNO3 forming a nitro-compound
In the results obtained, only the intact protein
and the acidic hydrolysate yielded a positive
result (clear yellow solution) while the basic and
enzymatic hydrolysates both resulted to a clear
colorless solution. From this, it can be said that
an aromatic amino acid (tyrosine or tryptophan)
is present on the terminal side of the protein
albumin in yielding a positive result for the intact
protein. It can also be said that this aromatic
amino acid was procured during the acidic
hydrolysis of the intact protein, while it is
possible that the amino acid was destroyed
during basic and enzymatic hydrolysis.
Table 3. Results of the Xanthoproteic Test
Xanthoproteic Test
- Clear white solution
Intact
- Clear yellow solution with
Protein
cloudy formation

Protein
Hydrolysate

Acidic

- Clear white solution

- Clear yellow solution

Basic
Enzymatic

Milky white solution

Clear colorless solution

Millons test is a test specific for tyrosine, the

only amino acid containing a phenol group, a
hydroxyl group attached to a benzene ring. In
Millons test, the phenol group of tyrosine is first
nitrated by nitric acid in the test solution. Then
the nitrated tyrosine complexes mercury(I) and
mercury(II) ions in the solution to form either a
red precipitate or a red solution, both positive
results [3].

Ninhydrin Test

Protein
Hydrolysate

Intact
Protein
Acidic
Basic
Enzymatic

Colorless solution
Colorless solution
Blue-violet solution
Clear colorless solution

Xanthoproteic test is a test for the presence of

aromatic amino acids which include tyrosine and
tryptophan, with phenylalanine not readily
nitrated and so does not respond to this test as it
is ordinarily performed. Aromatic amino acids
react
with
concentrated
HNO3
at
high
temperatures to form nitro-compounds which are
yellow in color. At alkaline pH, the color changes
to orange due to the ionization of the phenolic
group [7].

Figure 4. Millon Reaction Principle

Observations on the results obtained showed
that neither of the intact protein nor any of the
protein hydrolysates tested positive for this test,
with all results giving a clear white or cloudy
solution.
With this result, tyrosine can be eliminated
from the list of aromatic amino acids present in
albumin, leaving tryptophan as the aromatic
amino acid procured during the acidic hydrolysis
of the intact protein during the previously done
Xanthoproteic test.

Table 4. Results of the Millons Test

Protein
Hydrolysat
e

Millons Test
Intact Protein
Clear white
Acidic
Colorless clear solution
Basic
Cloudy white solution
Enzymatic
Clear white solution

The Hopkins-Cole test is specific for

tryptophan, the only amino acid containing an
indole group. As the protein solution is
hydrolyzed by the concentrated sulfuric acid at
the solution interface, the indole ring present in
tryptophan reacts with glyoxylic acid to form a
purple ring product [3]. Concentrated sulfuric
acid and insoluble water will not mix at once
which will result to the formation of two layers
with a purple ring on the surface of the solution
junction.

Figure 5. Purple ring formation from the reaction

of an indole ring in tryptophan with glyoxylic acid
The intact protein in this experiment was
observed to form a red ring at the junction where
it should have resulted to a purple ring. Positive
results are still recorded for the intact protein,
with a note indicating some errors in the
experiment that may have destroyed or lessened
the amino acid present in the sample. The protein
hydrolysates, on the other hand, presented
negative results, showing two layers of a clear
and cloudy solution. This means that tryptophan
is present at the terminal side of the intact
protein which may have been destroyed or
lessened during hydrolysis.
This result confirms the previously concluded
presence of tryptophan on the terminal side of
albumin; the presence of an aromatic amino acid
detected by the Xanthoproteic test and the amino
acid specified by both the Millon test and the
Hopkins-Cole test.
Table 5. Results of the Hopkins-Cole Test
Hopkins-Cole Test
Intact
Protein
Acidic
Protein
Hydrolysat
e

Basic

Enzymatic

Red ring at the junction

Clear colorless solution
2 layers (milky white at
junction)
- Cloudy white solution
- Clear colorless solution
with bubbles
Clear colorless solution

Sakaguchi test is a test for the amino acid

containing the guanidine group in Arginine [1].
The guanido (NH2-C=NH) of free arginine or

arginine residues in the protein reacts with Naphthol and sodium hypobromite to give a
bright red-orange colored complex [10].

Figure 6. Complex formation from guanidine

group reaction with -Naphthol and sodium
hypbromite.
Since alkaline or basic hydrolysis destroys
arginine, the basic hydrolysate presented a
negative result (clear light brown solution) while
the acidic hydrolysate presented a positive result
(orange solution). The negative result obtained
from the reaction with the intact protein
(colorless solution with a cloudy formation)
indicates that the arginine is a non-terminal
amino acid while the enzymatic hydrolysate
which had a clear light brown solution same to
that of the basic hydrolysate indicates that
arginine was also destroyed during enzymatic
hydrolysis.
Table 6. Results of the Sakaguchi Test

Protein
Hydrolysat
e

Sakaguchi Test
Colorless solution with
cloudy formation
Orange solution
Clearl light brown solution
Very clear light brown
Enzymatic
solution
Intact
Protein
Acidic
Basic

Nitroprusside test is specific for cysteine, the

amino acid containing a sulfhydryl group (-SH).
The group reacts with nitroprusside in alkaline
solution to yield a red complex [3]. The formation
of this red-purple colored complex is also called
the Mrner test [1].

Figure 7. Red complex formation from the

reaction of nitroprusside with cysteine.
Observations on the results obtained showed
that neither of the intact protein nor any of the
protein hydrolysates tested positive for this test,
with all results giving a clear yellow or dark
yellow-orange solution.
Table 7. Results of the Nitroprusside Test

Protein
Hydrolysat
e

Nitroprusside Test
Intact Protein
Clear yellow solution
Dark yellow-orange
Acidic
solution
Basic
Clear yellow solution
Enzymatic
Clear yellow solution

Fohls test is a test for sulfur-containing

proteins. It also indicates the presence of

methionine and cysteine which are amino acids

both containing sulfur in their structures. A
positive result for this test is the formation of
black precipitate from lead sulfide (PbS). The
dark coloration of the intact protein and the
acidic hydrolysate indicates the presence of sulfur
from these samples while the basic hydrolysate
presented a negative result which may indicate
that the amino acid present is methionine, as its
sulfur is not readily removed by alkali and is not
affected by this reaction.
Table 8. Results of the Fohls Test
Fohls Test

Protein
Hydrolysate

Intact
Protein
Acidic
Basic
Enzymati
c

Dark brown in color solution

Dark brown solution
Clear colorless solution
Clear light yellow solution

The test for amides indicates the presence of

primary, secondary and tertiary amides (in
asparagine and glutamine) and nitriles (in
arginine). A positive result for this test is the
change in color of litmus paper from red to blue,
as a result of the process of basic hydrolysis.
Basic hydrolysis causes partial or complete
decomposition of cysteine, arginine, and lysine.
The amide groups of glutamine and asparagine
release ammonia and the sulfur-containing amino
acid, cysteine, is partially destroyed to release
unoxidized sulfur in the hydrolysate [6].
All the protein hydrolysates gave a positive
result for this test, turning the litmus paper from
red to blue while the intact protein was observed
to give a neutral result with the red litmus paper
unchanged. This confirms the presence of
arginine (from Sakaguchi test) being a nonterminal amino acid present in albumin as the
intact protein presented negative results.
Table 9. Results for the Test for Amide

Protein
Hydrolysat
e

Test for Amide

Red red litmus paper
Intact Protein
Red blue litmus paper
Acidic
Red blue litmus paper
Basic
Clear light yellow solution
Enzymatic
(red blue litmus paper)

Pauly Test is specific for the detection of

tyrosine and histidine. The reagent used for this
test contains sulfanilic acid dissolved in
hydrochloric
acid.
Sulfanilic
acid
upon
diazotization in the presence of sodium nitrite
and hydrochloric acid results in the formation of a
diazonium salt. The diazonium salt formed
couples with the imidazole ring of histidine or the
phenol group of tyrosine in alkaline medium to
give a red-colored chromogen (azo dye) [7].
The reaction of the intact protein and the
protein hydrolysates observed using this test

showed negative results, all samples giving a

yellow-orange to orange solution. This further
confirms that tyrosine is not present in albumin
as was already observed from the negative
results of the Millon test.
Table 10. Results of the Pauly Test
Intact Protein
Acidic
Protein
Hydrolysat
e

Basic
Enzymatic

Pauly Test
Yellow orange solution
Yellow orange solution
Clear light orange
solution with white
precipitate
Clear orange solution

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