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, H2NCONHCONH2, 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 HNO3, 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 Figure 8. Structure of cysteine

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 HNO 3:
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
H2SO4 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% NaNO2 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% Na2CO3.
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

TESTS
Samples
NINHYDRIN BIURET
1. Glycine intense blue blue/light blue solution
2. Albumin purple solution purple solution
3. Gelatin purple solution purple solution
4. Distilled water colorless solution blue/light blue solution

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 Observation
1. Tryptophan blood red solution
2. Albumin orange solution
3. Distilled water 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 nitro-derivatives. 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 Observation
1. Gelatin 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
1. Tryptophan ring formed pink
2. Gelatin colorless, violet ring formed turbid white
violet ring formed at the junction of two fluids, light
3. Albumin turbid white
brown at top fluid
4. Distilled water colorless, no ring formed very light yellow
 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 (H2SO4in 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 H2SO4 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 hair dissolved; dark brown solution
4. Distilled water 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).

+ 2 + 2 + 2
2 + + 2
(3 )2 + 2 +23

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
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