=============

BIOORGANIC
CHEMISTRY
=============
BioOrganic Chemistry Laboratory CH205 (2015-2016) Experiment 8

Characterization of Proteins and Amino Acids through Color Reaction

Mary Bernardine G. Bagalay, Julie Ann Kim F. Berdonado,
Jeanne Isabelle B. Bilasano, and Assumpta Minette C. Burgos*
Department of Speech Language Pathology, College of Rehabilitation Sciences
University of Santo Tomas, Espana Street, Manila 1008
Date Submitted: April 5, 2016
Abstract:
Proteins are carbohydrates responsible for growth and cellular maintenance in the body. It is made up of amino acids linked
by peptide bonds. Identifying the amino acid content of a protein can be feasible through different test specific for an amino
acid. After conducting Biuret Test, Ninhydrin Test, Hopkins-Cole Test, Lead Acetate Test, Sakaguchi Test and
Xanthoproteic Test, the amino acid in albumin and gelatin were determined. Albumin is made up of tryptophan, cysteine,
methionine, arginine, tyrosine, and phenylalanine. On the other hand, gelatin is made up of arginine, tyrosine, tryptosan, and
phenylalanine
Keywords: proteins, amino acids, Biuret Test, Ninhydrin Test, Hopkins-Cole Test, Lead Acetate Test, Sakaguchi Test, and
Xanthoproteic Test.

Introduction
Biochemistry is the branch of science that tackles the chemical processes involved in living
organisms. It has three factors namely carbohydrates, lipids, and proteins. Carbohydrates are sources of
energy of ones body. These are sugars, starches, celluloses and gums that occur in living tissues and
food. Lipids is for energy reserve, signalling and serves as structural components of cell membranes.
Some examples of which are fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K),
monoglycerides, diglycerides, triglycerides, and phospholipids. On the other hand, proteins are the
bodys source of energy for growth and cellular maintenance.
Proteins are the second largest component of cell, next to water. It consists of amino acid linked
together by peptide bond. A peptide bond is formed when the carboxyl group of one molecule reacts
with the amino group of the other molecule. This is a dehydration synthesis reaction, also known as a
condensation reaction (Whitford, 2013). Amino acids are the building blocks of proteins. A typical
protein has amino folded into a 3-dimensional shape. Every protein has a unique shape and structure
which determines its function. If the shape unfolds, the protein will no longer function and will be
destroyed by the cell. This unfolding of the protein is called denaturation.

*Assumpta Minette C. Burgos

There are about 300 amino acids in nature, but only 20 are used to make proteins in the body.
Essential amino acids are required in diet and which humans are incapable of forming requisite. Among
the list of essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan, and valine. While, nonessential amino acids are not required in the diet namely
alanine,
asparagine,
aspartic
acid,
and
glutamic
acid.
When amino acids are tested on, it reacts due to its amphoteric nature and the R-group or side
chain. An amphoteric means it is capable of reacting to both acids and bases. The side chain or
functional groups present in amino acid determines the intensity of the product color. The reaction of a
sample under different tests can be a key point in determining the components of it.
The groups objective was to identify structural features common to amino acids by conducting
specific chemical tests to characterize the different amino acids. Thereafter, the group had to identify the
amino acids present in albumin, casein and gelatin using the principles of each chemical test as basis.

Methodology
The apparatus and materials used in this experiment depended on the test conducted by the group
and the groups number. Six chemical tests were conducted. These were Biuret test, Ninhydrin test,
Hopkins-Cole test, Lead Acetate test, Sakaguchi test, and Xanthoproteic test. Originally, there was
supposed to be another test, the Millons test however, Millons test involves mercury which can be
hazardous for the students. Hence, the professors decided to skip this test. Moreover, time constraints
led to the distribution of samples to each group. Odd groups were assigned to use albumin, while the
even groups on gelatin. In addition to the protein samples, each were given amino acid samples. Here is
the assigned sample to each group arranged in progressing group number: arginine, cysteine (protein),
glycine, histidine, methionine, phenylalanine, proline, serine, tryptophan, and tyrosine. Group 10 and 11
were assigned to tyrosine. Overall, 10 samples were involved in this experiment. Before the group
started, 10 drops of gelatin and another 10 drops cysteine were collected in different test tubes.
The tests were divided into general tests for proteins and amino acids and specific tests for amino
acids. The general tests for proteins were Biuret and Ninhydrin. In Biuret Test. 13 x 100 test tubes, 10%
sodium hydroxide (NaOH), and 0.01% copper (I) sulphate (CuSO4) was used this test. Then, 10 drops of
10% NaOH and 2 drops 1% CuSO4 were added to each of the test tubes. The test was mixed and the
color observed was noted. The other test, Ninhydrin test, required 13x100 test tubes, 0.1% Ninhydrin in
ethanol and a boiling water bath. Beaker halfway filled water was placed on a hot plate for boiling. After
adding 10 drops of 0.1% ethanolic ninhydrin to the samples, the test tubes were heating in a boiling bath
for two minutes. The test tubes were taken out and color observed was noted.
The specific tests for amino acids were Hopkins-Cole Test, Lead Acetate Test, Sakaguchi Test
and Xanthoproteic Test. 13 x 100 test tubes, Hopkins-Cole reagent and concentrated H2SO4 were needed
for Hopkins-Cole Test. 20 drops of Hopkins-Cole reagent were added to the prepared test tubes filled
with gelatin and cysteine and then mixed. The test tube was inclined at a 45. Then, 20 drops of
concentrated H2SO4 was carefully added into the solution. The color of the interphase was observed. It is
imperative to note that after adding concentrated H2SO4, shaking of the test tubes must not be made as it
may disrupt the process.
*Assumpta Minette C. Burgos

Next, Lead Acetate Test involves 13x100 test tubes, 40% NaOH (or concentrated NaOH), 1%
Pb(Ac)2 solution, and a boiling water bath. 10 drops of 40% NaOH (or concentreated NaOH) was added
to the prepared test tubes filled with gelatin and cysteine. After 5 minutes of heating in a boiling water
bath, 5 drops of 1% Pb(Ac)2. Some yielded with a precipitate while some did not. Nevertheless, the
color of the precipitate and/or the solution was observed and noted.
13x100 test tubes, 10% NaOH, 0.02% ethanolic -naphthol, and 2% NaOBr solution aided the
group for the Sakaguchi Test. 10 drops of 10% NaOH and 10 drops of 0.02% ethanolic -naphthol was
added to the solution. Then, it was mixed. After 3 minutes, 5 drops of 2% NaOBr solution followed. The
color of the solution was observed and noted.
The last test was Xanthoproteic Test. This test required 13x100 test tubes, concentrated HNO 3,
40% NaOH (or concentrated NaOH), a red litmus paper, and boiling water bath. 6 pieces of red litmus
paper was provided by the laboratory technician. Next, 10 drops of concentrated HNO 3 were added. The
group then heated the test tube. There was no required number of minutes for heating the test tube. But,
it is interesting to note that heating even for just 3 minutes would instigate a reaction with the nitric acid.
The group then gradually added 40%NaOH (or concentrated NaOH) dropwise until the solution turns
basic. They were able to establish that the solution was indeed basic after the red litmus paper turned
into blue after a drop of the solution. Both cysteine and gelatin turned basic after 20 drops of 40%NaOH
(or concentrated NaOH). The color of the solution was then observed and noted.
Observation and results of the different groups were then collected after the experiment.

Results and Discussion

TABLE 1. Results of Biuret Test
SAMPLE
OBSERVATION
1 Albumin
Purplish tinted clear solution
2 Gelatin
Clear light purple solution
3 Arginine
Light blue solution
4 Cysteine
Purple solution
(pale brown solution)
5 Glycine
Colorless
6 Histidine
Clear purple solution
7 Methionine
Colorless solution
8 Phenylalanine Light blue solution
9 Proline
Clear light blue solution
10 Serine
Light purple
11 Tryptophan
Clear solution with bluish tinge
12 Tyrosine
Clear light blue solution
The Biuret Test is a chemical test for detecting the presence of peptide bonds. If peptide bonds
were present, copper (II) ion forms violet-coordination complexes in alkaline solution (Mohanty &
Basu, 2006). The sole reagent, biuret reagent, in the Biuret Test for proteins contains hydrated copper
*Assumpta Minette C. Burgos

sulphate, potassium hydroxide solution, and potassium sodium tartrate. Hydrated copper sulphate
provides Cu (II) ions which forms the chelate complex. Chelate involves the formation or presence of
two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single
central atom. The Cu (II) also gives off the characteristic blue color of the reagent. Potassium hydroxide
solution does not directly participate with the reaction but is significant as it provides the alkaline
medium. Potassium sodium tartrate, on the other hand, stabilizes the chelate complex. As an alternative
reagent to the Biuret Test, sodium hydroxide, copper sulphate solutions and Fehlings Solutions A and B
may be used to produce comparable results.
Interestingly, biuret reagent is not named after the scientist who formulated Biuret Test but by
peptide bonds in biuret delivers a positive result for the test. However, the protein must have at least
three peptide bonds. A positive result yields a blue- to violet-colored solution. The intensity of the
color depends on the number of peptide bonds in the sample. Hence, the intensity of color is directly
proportional to the protein concentration in accord to the Beer-Lambert Law (David, 2001).

Albumin

Gelatin

Arginine

Cysteine

Glycine

Histidine

Methionine

Phenylalanine

*Assumpta Minette C. Burgos

Proline

Serine
Tryptophan
FIGURE 1.
Results of Biuret Test

Tyrosine

The result of the Biuret Test is expressed in Table 1 and can be observed in Figure 1. In the
experiment, all of the sample except glycine and methionine are expected to deliver a blue to purple
solution. This is because glycine, the simplest amino acid, does not have an amide link to which peptide
bonds can bind whereas, methionine is a free amino acid and does not contain a peptide bond. As
observed in Figure 1, albumin and gelatin had the most saturated purple hue. It can be inferred that these
two have the most peptide bonds and are proteins.
On the contrary, cysteine exhibited a pale brown solution. Although Biuret test repeated twice by
the group, it still exhibited the same pale brown color and this may be due to systematic error.
TABLE 2. Results of Ninhydrin Test
SAMPLE
OBSERVATION
1 Albumin
Indigo colored solution
2 Gelatin
Clear blue violet solution
3 Arginine
Colorless solution
4 Cysteine
Pink solution
(yellow solution)
5 Glycine
Dark violet solution
6 Histidine
Dark brown solution
7 Methionine
Transparent dark purple solution
8 Phenylalanine Dark violet solution
9 Proline
Dark red solution
10 Serine
Light orange solution
11 Tryptophan
Clear solution turned to apricot colored solution
12 Tyrosine
Golden yellow solution
The second test made by the group was the Ninhydrin Test. Ninhydrin Test is for detecting the
existence of amino acids. Ninhydrin is an oxidating agent that leads to deamination of alpha-amino acids
and proteins that contain free amino acids. It degrades amino acids into aldehydes, ammonia, and carbon
dioxide. The net result is a reduced form of hydrindantin. When ninhydrin condenses with ammonia,
hydrindantin produce a bluish-purple pigment known as the Rhuemanns purple. The Rhuemanns
*Assumpta Minette C. Burgos

purple is used to detect fingerprints due to the terminal amines or lysine residues. In addition to that,
ninhydrin also reacts with primary amines but the formation of carbon dioxide is quite problematic for
amino acids.

Albumin

Gelatin

Arginine

Cysteine

Glycine

Histidine

Methionine

Phenylalanine

Proline

Serine

Tryptophan
FIGURE 2.
Results of Ninhydrin Test

Tyrosine

Result of the Ninhyrdin Test is expressed in Table 2 and can be observed in Figure 2. The
expected positive visible result is a deep blue or purple solution with the exception of proline. Prolines
*Assumpta Minette C. Burgos

reaction is more yellow due to substitution of the alpha amino group that ninhydrin reacts with carbon
rings. Cysteine was expected to have a pinkish solution as result but exhbitied a clear yellow solution.
Again, the test was repeated by the group and yielded with the same result. Contamination of the test
tube could be considered as the cause of error.
TABLE 3. Results of Hopkins-Cole Test
SAMPLE
OBSERVATION
1 Albumin
Clear solution with purplish interphase
2 Gelatin
Slightly yellow
3 Arginine
Colorless interphase
4 Cysteine
Colorless solution
5 Glycine
Colorless
6 Histidine
Colorless
7 Methionine
Colorless solution
8 Phenylalanine Colorless solution
9 Proline
Clear colorless
10 Serine
Colorless
11 Tryptophan
Colorless solution with grayish interphase
12 Tyrosine
Colorless interphase
The third test is the Hopkins Cole Test. This test is used to determine the presence of amino
acid tryptophan. Tryptophan is the only amino acid containing an indole group. The
structure of an indole can be visualized in Figure 3. Hopkins-Cole reagent contains
glyoxylic acid (Mg powder, oxalic acid, and acetic acid) and concentrated H 2SO4.
The protein solution is hydrolysed by H2SO4 whereas reaction of glyoxylic acid
with a free tryptophan will yield a violet product.
Figure 3. Indole

Typtophan determines an indole nucleus and causes the formation of a violet

ring in the junction where the two layers meet- this implies a positive result. Some products do not show
the reaction, such as gelatin and zein. Hence, tryptophan in this experiment should exhibit a violet ring
in the junction where two layers meets.

Albumin

*Assumpta Minette C. Burgos

Gelatin

Arginine

Cysteine

Glycine

Proline

Histidine

Methionine

Serine
Tryptophan
FIGURE 4.
Results of Hopkins-Cole Test

Phenylalanine

Tyrosine

Result of the Hopkins-Cole Test is expressed in Table 3 and can be observed in Figure 4.
Albumin and tryptophan have distinct results from the rest of the samples who exhibited a colorless
solution. Tryptophan should have a violet interphase instead had a grayish interphase. Shaking of the
test tube or any action made on it could have disrupted the interphase formation. It is very critical that
the test tube should not be mixed or shook before observation as this interphase dispersion is fast.
Human error can be considered for this.

1
2
3
4
5
6
7
8
9

TABLE 4:
RESULTS OF LEAD ACETATE TEST
SAMPLE
OBSERVATION
Clear solution turned to clear brown solution
Albumin
Colorless solution
Gelatin
Colorless solution
Arginine
Murky solution
Cysteine
Colorless solution
Glycine
Light yellow solution without precipitate
Histidine
Colorless without precipitate
Methionine
Phenylalanine Colorless solution
Clear colorless solution
Proline

*Assumpta Minette C. Burgos

10 Serine
11 Tryptophan
12 Tyrosine

Clear light grayish tinted solution

Colorless solution
Colorless solution

Lead Acetate Test is used determining the presence of sulphur from cysteine and methionine.
Its reagent includes sodium hydroxide (NaOH) and lead (II) acetate. When NaOH is boiled, it converts
into S in the amino acid to NaS. Then, NaS then precipitates as balck PbS with the addition of lead (II)
acetate (Kulkarni, Rathod, Thonte, & Ghiware, 2008). To determine a positive result, solution should be
color black or a presence of a precipitate should be observed.

Albumin

Gelatin

Arginine

Cysteine

Glycine

Histidine

Methionine

Phenylalanine

*Assumpta Minette C. Burgos

Proline

Serine

Tryptophan

Tyrosine

FIGURE 5:
Results of Lead Acetate Test
The result of this test is presented in Table 4 and can be observed in Figure 5. Although albumin
exhibited a brown solution and not black, it can still be considered as a positive result.
TABLE 5: RESULTS OF SAKAGUCHI TEST
SAMPLE
OBSERVATION
1 Gelatin
Clear grayish solution turned to red solution
2 Albumin
Clear red orange solution
3 Arginine
Dark red solution
4 Cysteine
Saturated yellow solution
5 Glycine
Pale brown solution
6 Histidine
Clear brown solution
7 Methionine
Clear gold solution
8 Phenylalanine Cloudy light orange solution
9 Proline
Golden yellow solution
10 Serine
Light yellow solution
11 Tryptophan
Clear grayish turned to clear brownish solution
12 Tyrosine
Clear orange solution
The fifth test is Sakaguchi Test. Contrary to the Biuret Test, Sakaguchi Test acquired is named
after the scientist who first described it, Schoyo Sakaguchi. Sakaguchi reagent has 1-naphtanol and
sodium hypobromite. Under alkaline condition, - naphthol (1-hydroxy naphthalene) reacts with a
guanidine compound like arginine, which upon treatment with hypobromite or hypochlorite, produces a
characteristic red color through oxidation reaction. Another distinct characteristic of Sakaguchi is that it
is specific for arginine. Hence, samples with a positive result in Sakaguchi test is an arginine-containing
sample.

*Assumpta Minette C. Burgos

Albumin

Gelatin

Arginine

Cysteine

Glycine

Histidine

Methionine

Phenylalanine

Proline

Serine
Tryptophan
FIGURE 6.
Results of Sakaguchi Test

Tyrosine

The results of the Sakaguchi Test is seen in Table 5 and observed in Figure 6. Positive test
results are in an orange- to red-colored solution. In this test, albumin and gelatin generated a positive
result for this test.

*Assumpta Minette C. Burgos

1
2
3
4
5
6
7
8
9
10
11
12

TABLE 6. Results of Xanthoproteic Test

SAMPLE
OBSERVATION
Cloudy white solution turned to clear solution
Albumin
after water bath then clear yellow solution
After 20 drops NaOH clear neon yellow at 15
Gelatin
drops (basic)
Colorless solution
Arginine
Yellow solution
Cysteine
Colorless solution
Glycine
Colorless solution
Histidine
Colorless solution
Methionine
Phenylalanine Colorless solution
Clear colorless solution
Proline
Colorless solution
Serine
Yellowish solution turned to red solution after 20
Tryptophan
drops of NaOH
Rusty orange solution
Tyrosine

The last test involved in this experiment is the Xanthoproteic Test. It is used to determine the
presence of an activated benzene ring. Only amino acids that have activated benzene ring can undergo
nitration(Mohanty & Basu, 2006). Nitric acid gives color when heated with proteins containing tyrosine
and tryptophan. The results of this test are specific for tyrosine, tryptophan, and phenylalanine. Hence,
positive results would mean that the sample contains tyrosine, tryptophan, and phenylalanine. To
determine a positive result, a dark yellow solution should be perceived.
The result of Xanthoproteic Test is presented in Table 6 and can be observed in Figure 7. Only
albumin, gelatin, cysteine, and tryptophan have positive results. Moreover, tryptophan turned from
yellow solution to a red solution after 20 drops of NaOH. Lastly, tyrosine expressed a rusty orange
solution. The rusty orange color tyrosine indicates presence of an aromatic acid.

Albumin

*Assumpta Minette C. Burgos

Gelatin

Arginine

Cysteine

Glycine

Histidine

Proline

Serine

Methionine

Tryptophan
FIGURE 7.
Results of Xanthoproteic Test

Phenylalanine

Tyrosine

When amino acids are tested on it reacts due to its amphoteric nature and the R-group or side
chain. An amphoteric means it is capable of reacting to both acids and bases. The side chain or
functional groups present in amino acid determines the intensity of the product color. The color intensity
is dependent to the number of reacting functional groups. The greater the number, the more saturated the
color. Through the following tests conducted, the group was able to determine the amino acids present in
albumin and gelatin.
TABLE 8.
Results for Proteins
B
N
HC
LA
S
X

Albumin

X
X
Gelatin
*B for Biuret Test. N for Ninhydrin Test. HC for Hopkins-Cole Test.
LA for Lead Acetate Test. S for Sakaguchi Test. X for Xanthoproteic Test.
The table above shows as to what test did the protein garnered a positive result. The Biuret Test
and Ninhydrin Test confirm that albumin and gelatin have at least three peptide bonds and contain
amino acids. Hopkins-Cole implies that albumin contains tryptophan. Lead Acetate Test implies that
*Assumpta Minette C. Burgos

albumin contains cysteine and methionine. On the other hand, Sakaguchi and Xanthoproteic denote
presence of arginine and tyrosine, tryptophan and phenylalanine.
Conclusion
Proteins are made of amino acids. When amino acids are tested on, it reacts due to its amphoteric
nature and the R-group or side chain. An amphoteric means it is capable of reacting to both acids and
bases. The side chain or functional groups present in amino acid determines the intensity of the product
color. The greater number of functional groups that reacted, the more saturated its hue. Specific tests for
amino acid would express the amino acid content of a protein.

*Assumpta Minette C. Burgos

References
David, G. L. (2001). Analytical chemistry. India: University Press.
Kulkarni, M.V., Rathod, S. S., Thonte, S. S., & Ghiware, N. B. (2008). New Delhi: P ragati Books Pvt.
Ltd.

Mohanty, B & Basu, S. (2006). Fundamentals of practical clinical biochemisty. New Delhi: BI
Publications Pvt Ltd.
Whitford, D. (2013). Proteins: Structure and function. US: John Wiley & Sons

*Assumpta Minette C. Burgos