Ilar, Quenie Mariel

Date Performed:

BS-Chemistry

Date Submitted:

ASCHEM3
Activity 8
Color Reactions of Proteins and Amino Acids
Abstract: Amino acids are the building blocks of proteins. Each amino acid varies in
the side chains they possess which can be used as a tool in qualitative analysis in
proteins. The qualitative tests conducted include: Ninhydrin, Biuret, Xanthoproteic,
Millons, Hopmkins-Cole, Bromine water, Pauly, Reduced sulfur, and Sakaguchi
reactions. Albumin was confirmed to have the presence of tryptophan, tyrosine,
histidine, cysteine while gelatin only contains tyrosine, histidine, and arginine based
on the range of amino acids tested. Hair was also included in the Reduced Sulfur
test to check the presence of cysteine.Thus, amino acid derivatives are essential in
confirming the existence of certain amino acid in proteins through color reactions.
Introduction
Amino acids are critical to life since they have particularly important
functions like being the building blocks of proteins and being the intermediates in
metabolism. Amino acids possess an amine group, a carboxylic acid group and a
varying side chain that differs between different amino acids. The side chain
structure determines the class of the amino acid: nonpolar, neutral, acidic, or basic
(Milio and Loffredo, 1995).
Proteins (also known as polypeptides) are organic compounds made of amino
acids arranged in a linear chain. The amino acids in a polymer are joined together
by the peptide bonds between the carboxyl and the amino groups of adjacent amino
acid residues. Like other biological macromolecules such as polysaccharides and
nucleic acids, proteins are essential parts of organisms and participate in virtually
every process within cells (Nigam and Ayyagari, 2007).
Certain functional groups in amino acids and proteins can react to produce
characteristically colored products. The color intensity of the product formed by a
particular group varies among proteins in proportion to the number of reacting
functional, or free, groups present and their accessibility to the reagent (Milio and
Loffredo, 1995).
In this experiment, various color-producing reagents were used to
qualitatively detect the presence of certain functional groups in amino acids and
proteins. The principles of these qualitative tests are defined below.
Ninhydrin reaction

In the pH range of 4-8, all - amino acids react with ninhydrin

(triketohydrindenehydrate, shown in Figure 1), 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 proteins also react positively when subjected to the ninhydrin
reaction.
Figure 1. Structure of Ninhydrin

Biuret reaction
The biuret test for protein positively identifies the presence for proteins in
solution with a deep-violet color. Biuret, H 2NCONHCONH2, reacts with copper (II) ions
in basic solution to form a deep violet complex. The peptide linkages in proteins
resemble those in biuret and also form deep violet complexes with basic copper (II)
ion in solution. The general or biuret complex formed between the protein linkages
and the copper (II) ion of the biuret test is shown in Figure 2.

Figure 2. Structure of protein-copper(II) complex

Xanthoproteic reaction
Some amino acids contain aromatic groups that are derivatives of benzene.
These aromatic groups can undergo reactions that are characteristics of benzene
and 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. Figure 3 below shows the mechanism for Xanthoproteic reaction.

Figure 3. Xanthoproteic test reaction mechanism

Millons reaction
Millons test is specific to phenol containing structures (tyrosine is the only
common phenolic amino acid, Figure 4). Millons reagent is concentrated HNO 3, in
which mercury is dissolved. As a result of the reaction a red precipitate or a red
solution is considered as positive test.

Figure 4. Structure of Tyrosine

Hopkins-Cole reaction
This test is specific test for detecting tryptophan (Figure 5). The indole moiety
of tryptophan reacts with glyoxilic acid in the presence of concentrated sulphuric
acid to give a purple colored product.

Figure 5. Structure of Tryptophan

Bromine water test

The addition of bromine water and n-amyl alcohol to a solution containing
free tryptophan results in the formation of a pinkish lavender or violet color in the
alcohol layer. In the presence of excess bromine water, the pink color disappears
and it may be masked by the color of the reagent.
Pauly reaction
This test is specific for the detection of Tryptophan or Histidine (Figure 6). The
reagent used for this test contains sulphanilic acid dissolved in hydrochloric acid.
Sulphanilic acid upon diazotization in the presence of sodium nitrite and
hydrochloric acid results in the formation a diazonium salt. The diazonium salt

formed couples with either tyrosine or histidine in alkaline medium to give a red
coloured chromogen (azo dye).

Figure 6. Structure of Histidine

Reduced Sulfur Test

Proteins containing sulfur (in cysteine and cystine) give a black deposit of
lead sulfide (PbS) when heated with lead acetate in alkaline medium. The structures
of cystine and cysteine are shown in Figures 7 and 8, respectively.

Figure 7. Structure of cystine

of cysteine

Figure 8. Structure

Sakaguchi reaction
The Sakaguchi reagent is used to test for a certain amino acid and proteins.
The amino acid that is detected in this test is arginine (Figure 9). Since arginine has
a guanidine group in its side chain, it gives a red color with -naphthol in the
presence of an oxidizing agent like bromine solution

Figure 9. Structure of arginine

Methodology
The following test samples were subjected to different qualitative tests to
detect the presence of certain functional groups in amino acids and proteins:
Tryptophan, Glycine, Albumin, Gelatin, Hair, and distilled water as blank sample.

The reagents used in conducting the qualitative tests were: n-amyl alcohol,
freshly prepared bromine water, 0.55 copper(II) sulfate, glyoxylic acid, 10% lead
acetate, Millons reagent, -naphthol, freshly prepared 0.2% Ninhydrin solution,
concentrated HNO3, solid sodium acetate, Na2CO3, 10% and 20% NaOH, freshly
prepared 0.5% NaNO2, sulfanillic acid, and concentrated H2SO4.
A. Ninhydrin Reaction
Each of the following samples was neutralized with NaOAc: Glycine,
Albumin, Gelatin, and distilled water. To one mL of each sample, 2-3 drops of
0.2% freshly prepared Ninhydrin solution was added and was boiled in a
water bath for more than 2 minutes. The heated mixture was allowed to cool
and the resulting color was noted.
B. Buret (Piotrowskis) Reaction
A mixture was prepared with 1mL of 10% NaOH, , 1-2 drops of 0.5%
CuSO4 and 1 mL of each of the following samples: Glycine, Albumin, Gelatin,
and distilled water.
C. Xanthoproteic Reaction
One mL of each of the following samples was added with 1 mL
concentrated HNO3: Tryptophan, Albumin, distilled water. Each mixture was
immersed in a boiling water bath for 5 minutes, cooled, and was made
alkaline with 20% NaOH. Color change was noted.
D. Millons Reaction
Millons reagent was added to 1mL of each sample: Gelatin, albumin,
Peptone, and distilled water. For 10 minutes, the mixture was heated in
boiling water bath and was cooled afterwards. To the mixture, 0.5 mL of 0.5%
NaNO2 was added and was gently warmed. The resulting color of the
precipitate and/or the solution was observed.
E. Hopkins-Cole Reaction
In separate test tubes, 1 mL of Gelatin, Albumin, Tryptophan, and
distilled water was placed. Glyoxylic acid reagent (0.5 mL) was added to each
sample. One mL of concentrated H 2SO4 was layered in every test tube with
different samples. Tryptophan was present if a violet ring appeared at the
junction of the two fluids after a few seconds.
F. Bromine Water Test
Tryptophan, Gelatin, Albumin and distilled water were the samples
obtained and 1 mL was poured in separate test tubes. Two drops of freshly
prepared bromine water was mixed in ever test tubes. With 1 mL n-amyl
alcohol was mixed and shaken until the color of the alcohol layer was
observed.
G. Pauly Reaction
One mL of cold sulfanic acid with 1 mL of cold 0.5% NaNO 2 was mixed
in separate test tubes. For 3 minutes, mixtures were cooled with ice and
shaken constantly. In separate test tubes with the mixtures, 1 mL of Gelatin

and Albumin was added and alkaline with 10% Na 2CO3. Color formed was
noted and blank test was performed for blank test.
H. Reduced Sulfur Test
In separate test tubes, 1 mL of Gelatin, Albumin, Hair and distilled
water was poured and added with 1 mL of 20% NaOH and 2 drops of 10%
Pb(OAc)2. With a marble, the mixtures were covered and boiled in a water
bath for few minutes. The solution darkens if cysteine was present; the color
was deepening into black if sufficient sulfur was present.
I. Sakaguchi Reaction
Two mL of 20% NaOH was mixed in separate test tubes with 1 mL
samples of Gelatin, Albumin and distilled water and added with 2 drops of
alpha-naphthol reagent. The solution was mixed and added 0.5 mL fresh
bromine. The color formed was noted.

Results and Discussion

Tables 1. Ninhydrin and Biuret Reactions
Samples

TESTS
NINHYDRIN

BIURET

1. Glycine

intense blue

blue/light blue solution

2. Albumin

purple solution

purple solution

3. Gelatin

purple solution

purple solution

colorless solution

blue/light blue solution

4. Distilled water

Ninhydrin test
Amino acids contain a free amino group and a free carboxylic acid group that
react together with ninhydrin to produce a colored product. When an amino group is
attached to the alpha carbon on the amino acids carbon chain, the amino groups
nitrogen atom is part of a blue-purple product as shown in Equation 1 below.
Proteins also contain free amino groups on the alpha-carbon and can react with
ninhydrin to produce a blue-purple product. From the data on the table, Glycine is
an amino acid therefore it yielded the Ruhemanns purple. Since both albumin and
gelatin are proteins, they also react with ninhydrin to form a purple product.

Equation 1. Mechanism for Ninhydrin test

Biuret test
This test is used to differentiate between proteins and amino acids. 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 shown in Figure 2 back
in the first page of this paper. Since two peptide bonds are at least required for the
formation of this complex, only proteins respond positively to this test.
The protein samples, Albumin and Gelatin, formed the purple-colored Biuret
complex. Glycine did not respond positively to this test since it is an amino acid.
Table 2. Xanthoproteic Reaction
Samples
1. Tryptophan
2. Albumin
3. Distilled water

Observation
blood red solution
orange solution
colorless solution

Aromatic amino acids respond to this test. In the presence of concentrated

nitric acid, the aromatic phenyl ring is nitrated to give yellow colored nitroderivatives. At alkaline pH, the color changes to orange due to the ionization of the
phenolic group.
Tryptophan, an aromatic amino acid, gives a blood red solution because there
is partial oxidation of the substance by nitric acid. Albumin was observed to produce
an orange solution after the reaction since it is a protein which contains an aromatic
amino acid in its chain.
Table 3. Millons Reaction
Samples
1. Gelatin

Observation
deep red precipitate

2. Albumin

light pink precipitate

3. Distilled water

colorless solution

Millon's reagent gives positive results with proteins containing the phenolic
amino acid tyrosine.In this 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 a red precipitate or a red solution, both
positive results. Proteins that contain tyrosine will therefore yield a positive result.
However, some proteins containing tyrosine initially forms a white precipitate that
turns red when heated, while others form a red solution immediately (Milio and
Loffredo, 1995).
From the observed results in Table 3, both protein samples give a positive
result thus confirming the presence of tyrosine.
Table 4. Hopkins-Cole and Bromine Water test
TESTS

Samples

Hopkins-Cole

Bromine Water

ring formed

pink

2. Gelatin

colorless, violet ring formed

turbid white

3. Albumin

violet ring formed at the junction of two

fluids, light brown at top fluid

turbid white

colorless, no ring formed

very light yellow

1. Tryptophan

4. Distilled water

These tests are specific for tryptophan, the only amino acid containing an
indole group.
In the Hopkins-Cole reaction, the indole ring reacts with glyoxylic acid in the
presence of a strong acid (H 2SO4in the experiments case) to form a violet cyclic
product. The mechanism for this reaction is shown in Equation 2 below.
The Hopkins-Cole test confirms the presence of tryptophan in Albumin. The
protein solution is hydrolyzed by the concentrated H 2SO4 at the solution interface.
Once the tryptophan is free, it reacts with the glyoxylic acid to form the violet
product.
However, the results for the two qualitative tests were questionable. In the
case of Gelatin, according to Cole (2000), the protein lacks tryptophan which does
not agree to the results obtained in Hopkins-Cole test. In addition, albumin contains
tryptophan but showed negative results towards bromine water test.

Equation 2. Reaction Mechanism for Hopkins-Cole test

Table 5. Pauly Reaction

Samples

Observation

1. Gelatin

intense yellow orange

2. Albumin

intense yellow orange

3. Distilled water

light brown

The basic principle in pauly's test is diazotization (Equation 3). Sulfanilic acid
will be diazotized with the addition of sodium nitrite and sodium carbonate to form
diazonium component. Diazonium component react with the imidazole ring of
histidine and a phenol group of tyrosine to form dark red compound.
Gelatin and albumin both yielded intense yellow orange products which is still
considered as a positive result. The test confirms the presence of tyrosine, histidine,
or both. According to Cole (2000), Gelatin contains less than 1% of histidine and less
than 0.5% of tyrosine.

Equation 3. Diazotization of sulfanilic acid

Table 6. Reduced Sulfur Reaction

Samples

Observation

1. Gelatin

colorless

2. Albumin

dark brown solution

3. Hair
4. Distilled water

hair dissolved; dark brown solution

colorless

Sulphur containing amino acids, such as cysteine and cysteine, upon boiling
with sodium hydroxide, yield sodium sulphide. This reaction is due to partial
conversion of the organic sulphur to inorganic sulphide, which can be detected by
precipitating it to lead sulphide, using lead acetate solution. The balance equation
for this reaction is on Equation 4.
Among the three protein samples: gelatin, albumin, and hair, only gelatin
responded negatively to the test. According to Galewska et al. (2013), gelatin
(denatured collagen) does not contain cysteine while albumin contains all the
protein-building amino acids. Hair is composed of keratin which is a combination of

18 amino acids including cysteine. Cysteine, being rich in sulphur, plays an

important role in the cohesion of the hair (Beveridge & Lucas,1944).

RSH +2 NaOH ROH + Na 2 S + H 2 O

2
++ S
Na2 S Na

CH 3 COO

2 PbS+2 CH 3 COO

Pb
Equation 4. Balance equation for Reduced Sulfur reaction

Table 7. Sakaguchi
Reaction
Samples

Observation

1. Gelatin

reddish color solution

2. Albumin

reddish color solution

3. Distilled water

deep green solution

This test is specific for arginine, as it is the only amino acid containing a
guanidine group. This moiety reacts with -naphthol and an oxidizing agent
(bromine water in the experiments case) to give the red-colored complex.
Gelatin and Albumin was confirmed to have the presence of arginine from the
results obtained. Gelatin is said to have 8% of arginine (Cole, 2000).
Conclusion
Amino acids are building blocks of proteins. They possess an amine group, a
carboxylic acid group and a varying side chain that differs between different amino
acids. These side chains are essential in determining the presence of amino acids in
proteins through qualitative test. The qualitative tests conducted in this experiment
make use of various color-producing reagents which is dependent upon the side
chains present in the test samples. Ninhydrin reaction was used to confirm the
sample as an amino acid or protein. Biuret test was significant in distinguishing
proteins from amino acids. Xanthoproteic reaction is for classifying amino acids with
benzene derivative. Millons test was used to check the presence of tyrosine in the
test samples. Both Hopkins-Cole and Bromine water test are important for
tryptophan determination. Pauly reaction confirms the existence of tyrosine and
histidine in the sample. Reduced Sulfur test makes use of the sulphur in determining

the presence of cysteine and cysteine. And Sakaguchis reaction is useful in arginine
determination.
Albumin was confirmed to have the presence of tryptophan, tyrosine,
histidine, cysteine, and arginine. While for gelatin, only the existence of tyrosine,
histidine, and arginine were observed. Hair was also included in the Reduced Sulfur
test to check the presence of cysteine.
References
Beveridge J. M. R. and Lucas C. C. (1944). The analysis of hair keratin.Journal on
Biochemistry. 38(1): 88 95.
Cole, CGB. Francis, F.J., editor. (2000). Gelatin.. Encyclopedia of Food Science and
Technology, 2nd ed. 4
Vols. New York: John Wiley & Sons, pp. 1183-1188.
Galewska Z., Gogiel T., Makowski A., Romanowicz L., Sobolewski K., Wolaska M.
(2013). Biochemistry
Workbook. Medical University of Biaystok.
Milio, F. & Loffredo, W. (1995). Qualitative testing for amino acids and proteins. USA:
Chemical
Education Resources, Inc.
Nigam, A & Ayyagari, A. (2007). Lab Manual in Biochemistry: Immunology and
Biotechnology. New Delhi:
Tata McGraw-Hill Education. ISBN: 9780070617674
vlab.amrita.edu,. (2011). Qualitative Analysis of Amino Acid. Retrieved 15 January
2016, from vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1