PROTEIN TESTS

This laboratory report aims to discuss (6) tests for proteins as follows:
(A) Ninhydrin Test
(B) Biuret Test
(C) Xanthoproteic Test
(D) Millon's Test
(E) Hopkins-Cole Test
(F) Nitroprusside Test

A. Ninhydrin Test

Objectives:

● To detect the presence of proteins by performing Ninhydrin test.

● To discuss the principle and concept behind Ninhydrin test as a qualitative test for the
presence of amino acids
● To understand the clinical relevance of Ninhydrin test as applied in real-life clinical setup.

Reagents:

● 1% amino acid solution (standard solution)

● 2% Ninhydrin reagent (dissolved in acetone)
● Distilled water

Procedures:

● Prepare all the materials needed.

● Take 1 mL standard solution in dry test tube and 1 mL distilled water in another tube as
a control.
● Label the first test tube containing the standard solution as Test Tube #1 and the other,
Test Tube #2.
● Using a pipette, pour few drops (3-5 drops) of 2% Ninhydrin in both the test tubes.
● Place the test tubes in the water bath for 5 minutes and then allow cooling to room
temperature.
● Observe the formation of color and note down the result. (Note: The appearance of blue
or violet color indicates the presence of protein in the solution.)

Conclusion:
 Ninhydrin test is a general test for proteins and is used to detect the presence of any
amino acids in unknown samples. The reaction exhibits a positive result when the sample
contains amines or alpha-amino acids, indicated by a color change of the solution to a deep
blue color, which is also known as Ruhemann’s purple, which will be observed in the
experiment. Moreover, as seen on Figure 1, the Ninhydrin test exhibits different colors when
amino acids aside from amines and α-amino acid/s. In solutions containing ammonia, a
primary/secondary amine, or any α-amino acid exhibits a Purple (Ruhemman’s purple) color
change, while a solution with proline and hydroxyproline results to a yellow color, and lastly, for
proteins with a free amide group like asparagine, the color changes to brown.

Figure 1.
Color changes observed in positive Ninhydrin Test

In the experiment, two test tubes were prepared where one contains the 1% amino acid
solution (Test Tube #1) and the other tube contains distilled water (Test Tube #2) as seen in
Table 1. The distilled water serves as the negative control in the experiment to observe any
color change in the test tube containing the amino acid solution. As the 2% Ninhydrin is added
to each test tube, the test tube containing the amino acid solution exhibited a color change to a
purple-colored complex, indicating a positive Ninhydrin’s Test. This is due to the reaction
between the amino group of the free amino acid and the ninhydrin. Since ninhydrin is an
oxidizing agent, it results in the amino acid to undergo the process of deamination-- liberating
ammonia and carbon dioxide gases, along with an aldehyde and a reduced form of ninhydrin,
also known as hydrindantin. The ammonia liberated then continues to react with another
ninhydrin molecule to form a diketohydrin, which is responsible for the deep blue color also
known as the Ruhemann’s complex.

Test Tube Substances Present Observation

 (Color Change)

Test tube #1 1% amino acid solution Purple

(standard solution)

Test tube #2 Distilled water Color change is not evident.

(control)

Table 1.
Color Change Observation in the Sample Solution and Control

Overall, aside from being a general test for the detection of proteins, the Ninhydrin test is
often used to detect fingerprints because ninhydrin reacts with the terminal amines of lysine
residues in peptides and proteins shed off in fingerprints. In the field of medical laboratory
science, it may be used as a qualitative method to identify proteins and amino acids in
diagnostic laboratory tests, such as in the analyses of musculoskeletal diseases including
rheumatoid arthritis, tendinitis, osteoarthritis, and more. It is also applied in the fields of
biomedicine, food science, forensics, and agriculture.

B. Biuret Test

Objectives:

● To understand, expound and explain the principle behind the Biuret test
● To test for the presence of proteins in common food samples
● To demonstrate the role of the presence of the peptide bond in accordance to the Biuret
test
● To apply and relate the Biuret test in varying real-life applications

Reagents/Materials:

● 0.5 g Sodium hydroxide (NaOH flakes)

● 0.8 g Copper sulfate (CuSO4 powder)
● Water
● Egg white
● Milk
● Mashed banana
● Salt solution

Procedures:

● Preparation of NaOH solution

a. Take 0.5 grams of NaOH and put it in a container or bottle with a small opening.
 b. Fill the container with 30ml of water using the dropper.
c. Shake well to dissolve the flakes.
● Preparation of CuSO4 solution
a. Take 0.8 grams of CuSO4 crystals and transfer it in a small container or bottle
with a small opening.
b. Fill the container with 10mL of water and close it.
c. Shake well to dissolve the crystals
d. Now we have a diluted CuSO4 solution in the container.
● Preparation of Samples
1. 30 drops of the diluted NaOH and 5-6 drops of the CuSO4 solution was added to each
test tube containing milk, salt solution, egg white, and mashed banana

Conclusion:

The biuret test is used to determine the presence of peptide bonds in substances.
Peptide bonds link together amino acids, which are the constituents of proteins, therefore, the
Biuret test is also a general test for the detection of proteins in sample solutions or substances.
After adding the diluted NaOH and diluted CuSO4 solution into each of the four test tubes
containing milk, salt solution, egg white, and mashed banana, different color reactions were
observed (Table 2).

Test Tubes Food Sample Color Reaction

Test Tube #1 Milk Light purple

Test Tube #2 Salt solution Light blue

Test Tube #3 Egg white Dark purple

Test Tube #4 Mashed Banana Dark blue

Table 2.
Color reaction observed in each test tubes

In the Biuret test, a positive test result exhibits a purple color, and its intensity is
dependent on the length of the peptide chains, or the number of the peptide bonds present in
the sample. Egg whites and milk produced a color change to purple, which is indicative of the
presence of protein in these substances. Meanwhile, a negative test result shows a blue color,
as seen in the salt solution and mashed banana meaning that these substances do not contain
any protein (Figure 2). The reagents used in the test include a strong base (NaOH) and copper
(II) sulfate diluted in water. The alkali (NaOH) maintains the stability of the cupric ions in the
solution since the copper is responsible for reacting with the proteins to establish a purple-
colored complex.
 Figure 2.
Positive and Negative Result in Biuret Test

Ultimately, the Biuret test may also be applied in real-life, including the determination of
protein concentration in a sample (e.g. urine), since the intensity of the colored complex
produced in the positive test result has a direct correlation to the amount of peptide bonds
present. A more intense color is indicative of more peptide bonds, and consequently, since
peptide bonds occur with the same frequency per amino acid, it also indicates a higher protein
concentration. And likewise, a paler purple color would be indicative of lesser protein
concentration in the sample.

C. Xanthoproteic Test

Objectives:

● To differentiate aromatic amino acids which give positive result from other amino acids
● To detect the presence of protein in the given solution
● To detect the presence of aromatic groups-containing amino acids like tyrosine and
tryptophan

Reagents:

● 2 ml of protein solution
● 1 ml of concentrated nitric acid
● 40% NaOH
● Red litmus paper

Procedure:

1. Take 2 ml of protein solution in a test tube

 2. Add 1 ml of concentrated nitric acid
3. Heat the solution thoroughly and boil it for 1 minute
4. Observe the change of color in the test tube
5. Cool it under tap water
6. Divide the contents of the test tube into two parts
7. To one part, add 40% NaOH until the solution becomes alkaline.
8. Use red litmus paper to detect if it is alkaline
9. Observe the change in color
10. Compare with a negative test result in another test tube

Conclusion:

The Xanthoproteic test is a specific biochemical test for the detection of amino acids
containing only aromatic groups such as tyrosine and tryptophan in a sample solution. It exhibits
a type of nitration reaction in which the 1 mL concentrated nitric acid reacts with aromatic amino
acids such as tyrosine and tryptophan in the presence of heat. This reaction yields the formation
of a Xanthoprotein, which is yellow in color. Therefore, a positive test result for a Xanthoproteic
test exhibits a color change to yellow indicative of the presence of an aromatic amino acid, while
a negative test does not exhibit color change (Table 3). The color change is further amplified
through the addition of an alkali (NaOH), which turns the obtained color darker, from yellow to
orange due to ionization.

Positive Result Negative Result

Table 3.
Comparison of Positive and Negative result in Xanthoproteic test

There exists another amino acid that contains an aromatic group which is phenylalanine.
However, phenylalanine exhibits a very weak or negative result in the Xanthoproteic test due to
its highly stable phenyl group, which is therefore unable to react completely with the nitric acid
(HNO3) to produce a yellow complex (Figure 3). Overall, the Xanthoproteic test, being
responsible for the specific detection of aromatic amino acids only, may be used not only for the
general detection of proteins and amino acids but also in differentiating tryptophan and tyrosine
in a sample from other non-aromatic amino acids.
 Figure 3.
High stability of phenyl group in phenylalanine yields negative Xanthoproteic test result

D. Millon’s Test

Objectives:

● To define the concept of Millon’s test

● To identify the uses of the test how its reagent affects the sample
● To demonstrate the positive and negative Millon’s test reactions using the given samples
● To distinguish the positive samples and negative samples after mixing with the Millon’s
reagent
● To observe why some of the samples reacted to the reagent and otherwise
● To apply and relate Millon’s test in everyday life

Reagents:

● Millon’s reagent
● 1% Phenol solution
● 1% Tyrosine solution
● Egg albumen solution
● 1% Starch solution
● Control sample of Distilled Water

Procedures:

1. Put the 5 labeled test tubes on the test tube stand

 2. Using different droppers or pipettes, add 1mL or 2 mL of 1% phenol, 1% tyrosine, 1%
starch, egg albumin, and distilled water to each test tubes. The test tube containing
distilled water will serve as the negative control for this experiment.
3. Using a different dropper or pipette, add 2 mL of Millon’s reagent to the test tubes.
Formation of white precipitate can be observed usually after addition of Millon’s reagent,
but this is not the end result.
4. Mix the contents of the tubes by gently swirling. If no color changes occur after a few
minutes, tubes may be placed in a hot water bath for 1 to 2 minutes.
5. Observe color formation (brick red or pink color).
6. Record the positive and negative Millon’s test.

Conclusion:

The Millon’s test is a specific protein test for the detection of the phenol group in
samples, which is exclusively found in the amino acid tyrosine. The formation of a color change
in the solution into a brick-red or pinkish color denotes a positive Millon’s test result (Figure 4).
In the experiment, the test tubes which contain phenol, tyrosine, and egg albumin resulted in
this color change to a pinkish and brick-red color, which means that these substances contain
tyrosine. In contrast, the absence of a color change, as seen in the test tubes containing the
starch and the distilled water.

Figure 4.
Brick-Red product formation in Positive Millon’s test

After adding the Millon’s reagent, a color change to a brick-red color was observed in the
test tubes containing the phenol, tyrosine, and egg albumin (Table 4). This is specifically due to
the phenol group, which is exclusively found in tyrosine among all the amino acids, to undergo
the process of nitrification in the presence of the Millon’s reagent, which is composed of
mercuric and mercurous nitrates dissolved in strong nitric acid, to form complexes of with heavy
metals such as mercury. Overall, this reaction of the phenol group with the Millon’s reagent
forms mercuric fumarate which gives the pinkish or red coloured compound. This is why the
phenol and tyrosine-containing test tube may serve as the positive control of the Millon’s test,
and since the egg white albumin exhibited the same color change as these tubes and indicated
a positive Millon’s test, it can be concluded that the egg white albumin contains tyrosine, and
specifically, a phenol group. Meanwhile, the distilled water serves as the negative test control in
the experiment which did not exhibit any color change. Since the starch in the test tube did not
have any color change to a brick-red color and remained clear (Table 4), starch does not
contain any phenol group nor any tyrosine amino acid. Ultimately, since tyrosine is present in
the structure of the majority of proteins, it may also be used as a general test for proteins in
samples. However, due to its specificity for the phenol group, it may also be used to detect this
functional group in other non-protein samples, which makes it an unreliable general test for
proteins only in a sample.

Test Tube Reagents Color Results Final Results

1 Phenol Pink to light red Positive Millon’s test

2 Tyrosine Brick red Positive Millon’s test

3 Egg Albumin Light red with white Positive Millon’s test

precipitate

4 Starch Clear Negative Millon’s test

5 Distilled water Clear Negative Millon’s test

(control)

Table 4.
Summary of results in each test tube for Millon’s Test

E. Hopkins-Cole Test (Adamkiewicz Reaction)

Objectives

● To become knowledgeable about the concepts and principles of Hopkins-Cole test

● To determine the applications and uses of Hopkins-Cole test
● To detect the presence of indole ring containing amino acid in proteins.
● To detect the presence of tryptophan-containing proteins.

Reagents:

● Hopkin’s Cole reagent: Glyoxylic acid- It can be prepared by exposing glacial acetic acid
to sunlight for a few days.
● Concentrated H2SO4
 ● Sample: 0.1% amino acid solution
● Negative control: Distilled water

Procedure

1. Prepare all the reagents and materials needed.

2. In a test tube, 2 ml of glyoxylic acid or light-exposed glacial acetic acid and 2 ml of the
sample liquid are combined.
3. To this, concentrated H2SO4 is added along the sides of the test tube held at a slanting
position. Two distinct layers of liquid are to be formed without mixing.
4. The test tube should be observed for the formation of a colored ring at the interface of
two layers.

Conclusion:

The Hopkins-Cole test, also known as the Adamkiewicz reaction, is a specific test for the
detection of the indole ring present in tryptophan amino acids in proteins. It exhibits the
formation of a purple-colored ring when tryptophan is present in a protein solution. In contrast,
no formation of a purple ring is evident in a negative result, when there are no tryptophan amino
acids present in a solution. This formation of a colored ring is due to the layering of the
concentrated sulfuric acid (H2SO4) that was added into the test tube containing the Hopkins
Cole reagent (glyoxylic acid). The indole group present in the amino acid tryptophan reacts with
glyoxylic acid in the presence of the concentrated sulfuric acid (H2SO4) to give a purple-colored
complex. The H2SO4 added to the reagent helps to stabilize the Hopkins Cole reagent and
prevent its decomposition and the release of carbon dioxide. The reaction is illustrated in Figure 5
below.

Figure 5.
Reaction of Tryptophan and glyoxylic acid in the presence of H2SO4 producing a purple-colored
product

In addition, the indole group that is present in the molecular structure of tryptophan is the
determinant of whether the Hopkins Cole test will exhibit a positive or negative result. Other types of
amino acids will give a negative result to the test since an indole group is exclusively present in the
tryptophan among all amino acids. As the aldehyde group from the glyoxylic acid reacts with this
indole group from the tryptophan, a chemical reaction occurs. The reaction exhibited by the indole
ring in the tryptophan is known as a condensation or dehydration reaction, which resulted in a
colorful condensation product, along with one molecule of water, indicative of the formation of the
purple ring in the test tube.

F. Nitroprusside Test
Objectives

● To detect the presence of amino acid cysteine in a protein solution

● To observe and analyze the color change of the protein solution a containing thiol group
in negative and positive test results
● To explain the role of ammonium hydroxide (alkali) in the detection of amino acid
cysteine
● To identify the other uses of the nitroprusside test in the clinical and laboratory setting
● To differentiate between the amino acid cysteine and cystine

Reagents:

● Sulphur amino acids (1.0% cystine, and methionine)

● Sodium nitroprusside (2% prepared fresh)
● Ammonium hydroxide

Procedures:

1. Transfer 2 mL of amino acid (sample) solution

2. Add 0.5 mL nitroprusside solution and 0.5 mL ammonium hydroxide and shake
thoroughly.
3. Heat the test tube over a heating lamp
4. Observe the color change

Conclusion:

The nitroprusside test is a very specific protein test for the amino acid cysteine only,
which has a thiol or sulfhydryl functional group in its structure that reacts with nitroprusside to
exhibit a red complex. When a protein solution contains cysteine, it exhibits a color change from
clear (transparent) to a red color (Table 5) thus indicating a positive result in the nitroprusside
test. However, when a protein solution does not contain cysteine, it will remain clear and no
color change to red will be observed, indicating a negative result (Table 5). This occurs due to
the reaction between the nitroprusside reagent and the free -SH or specifically the sulfur atom in
the amino acid cysteine, in the presence of an alkali (ammonia). This reaction ultimately results
in a red complex as shown in the reaction formula below:
 Negative Control Sample Positive Control

Table 5.
Comparison of the sample with the positive and negative test controls

Cysteine is not the only amino acid which has a sulfur atom, since methionine also
contains a sulfur atom in its structure (Figure 6). However, the nitroprusside test still remains
specific for the detection of cysteine since it exhibits a negative result for methionine.
Methionine has a strong thioester linkage present in its sulfur atom, thus making it difficult to
detach from its structure and is therefore unable to react with the nitroprusside reagent (Figure
6).

Figure 6.
Presence of sulfur atom in both cysteine and methionine
 Moreover, the reason behind why an alkali, specifically ammonium hydroxide or
ammonia was present in the experiment is due to its role of detaching the sulfur atom from
cysteine, therefore making it more accessible to react with the nitroprusside and exhibit a red
color change. However, many protein solutions do not readily exhibit a color change even with
the addition of an alkali. In these instances, heat is applied to the solution in order to promote
heat coagulation, which may further be necessary for the complete detachment of thiol groups
or sulfur atoms from the cysteine amino acid molecules, as done in the experiment.

Apart from the detection of cysteine in protein solutions, the nitroprusside test may also
be utilized in the clinical and laboratory settings, primarily in the detection of compounds such
as ketones mostly tested for urine samples and in diagnosis for cystinuria, which is an inherited
metabolic disorder characterized by excessive amounts of undissolved cystine in the urine. It
can also be used for the differentiation of between cysteine and cystine, as cysteine (Cys) gives
a positive result to this test, while cystine exhibits a negative result. Although similar in their
diction, these two molecules are different in their molecular structure (Figure 7) and
consequently, different in their functions and characteristics. Cysteine is a sulfur-containing
amino acid while cystine forms when these two amino acids join via a disulfide bond, making it
less readily soluble in the body

Molecular Structure of Cysteine Molecular Structure of Cystine

Figure 7.
Comparison of the Molecular Structure of Cysteine and Cystine
.