Ana Liza D.

Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

I. INTRODUCTION
Proteins, essential constituents of the living cells, are high molecular weight compounds
which upon hydrolysis produce amino acids. In a protein, the amino acids are linked by peptide
bonds.
A variety of tests exist to determine whether a substance or a solution contains proteins or
protein breakdown products (e.g. proteoses, peptones, polypeptides, and in some tests, amino
acid). These depend on the presence of certain functional groups which react with a specific
reagent to produce a certain change in color, odor, and/or solubility.

COLOR REACTIONS OF AMINO ACIDS

GENERAL TESTS
1. Biuret Test
It is a general test for proteins comparable to molisch’s test for carbohydrates. A
violet color appears when Biuret’s reagent (0.1% CuSO 4 + NaOH) is added to any
compound which contains 2 or more of the following groups which all contain the – CO – NH2
groups.

2. Ninhydrin Test
In the presence of strong alkalies and of amino acid, beta-naphthoquinone sulfonic
acid develops a striking, deep red color. However, urea, uric acid, creatinine, and hippuric acid do
not yield colors.
Shows positive test for:
Alpha amino acids and proteins that contain free amino acid groups .

A positive test is indicated by:

The formation of red, blue or purple color indicates a positive result.

A negative test (left) and a positive A ninhydrin test is used to detect fingerprints.

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City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

test (right) Picture Source: libretexts.org

3. Folin’s Reagent
Folin's reagent or sodium 1,2-naphthoquinone-4-sulfonate is a chemical reagent used as a
derivatizing agent to measure levels of amines and amino acids. The reagent reacts with them in
alkaline solution to produce a fluorescent material that can be easily detected.

SPECIFIC TESTS
4. Millon’s Test
This a test for the presence of a phenolic ring, as in tyrosine which gives a red color with
Millon’s reagent made by dissolving mercury in nitric acid. The test was developed by the
French chemist Auguste Nicolas Eugene Millon (1812–1867). The reagent is made by dissolving
metallic mercury in nitrous acid and diluting with water. In the test, the phenol group in the side
chain of tyrosine gets nitrated, and that product then complexes with Hg(I) or Hg(II) ions to give
red coloration or precipitate. Millon's test is not specific for proteins; it also gives a positive test
for other compounds containing the phenol functional group.

5. Xanthoproteic
It detects the presence of benzene rings on which there are amino groups (e.g. tryptophan)
or hydroxyl groups (e.g. tyrosine) which are easily nitrated to give yellow colored aromatic nitro
compounds (xanthoproteic acid).
Xanthoproteic test is used to detect amino acids containing an aromatic nucleus (tyrosine,
tryptophan and phenylalanine) in a protein solution which gives yellow color nitro derivatives
on heating with conc. HNO 3. The aromatic benzene ring undergoes nitration to give yellow
colored product. Phenylalanine gives negative or weakly positive reaction though this amino
acid contains aromatic nucleus because it is difficult to nitrate under normal condition. On
adding alkali to these nitro derivative salts, the color change from yellow to orange.

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City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

6. Hopkins-Cole Test/Tryptophan Reaction

When tryptophan or containing tryptophan is heated with strong sulphuric or hydrochloric
acid in the presence of aldehyde, a blue or violet color is produced. The color is due to a
condensation of the alpha-hydrogen of the indole nucleus with an aldehyde.
The test is known under a variety of names, depending on the aldehyde employed. Many
proteins give the test without the addition of an aldehyde, owing either to contaminations or to
the presence of carbohydrate units in the protein molecule. When carried out in this way the test
is known as the Liebermann reaction. When formaldehyde is added, the reaction carries the
name of Acree-Rosenheim. In the Adamkiewicz reaction, glacial acetic is employed because it
contains glyoxalic acid, COOH-CHO. In the Ehrlich reaction, p-dimethylaminobenzaldehyde is
used; Cole’s test employs the unsubstituted benzaldehyde; and the Hopkin’s-Cole Test uses
performed glyoxalic acid.

http://biocheminfo.com/2020/04/05/hopkins-cole-test-adamkiewicz-hopkins-test-principle-reaction-reagents-procedure-and-result-interpretation/

7. Reduced Sulfur Test

It is a test for sulfur - containing amino acids cysteine and cystine (except for methionine
where its sulfur is not reactive), which react with acetate to form black lead sulfide.

Expected Results

 Positive: Darkening of the medium (a black precipitate) or blackening of the line of

inoculation indicates the presence of bacteria producing hydrogen sulfide.
 Negative: A negative H2S test is denoted by the absence of blackening.

3 | NOTRE DAME OF MARBEL UNIVERSITY

City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

8. Sullivan Cystine Reaction

The Sullivan reaction is a chemical test used for detecting the presence of cysteine or
cystine in proteins. A red colour appears when a protein with cysteine or cystine is heated with
sodium 1,2-naphthoquinone-4-sulfonate (Folin's Reagent) and sodium dithionite under alkaline
conditions. This was pioneered by the American Organic and Industrial Chemist, Eugene
Cornelius Sullivan (1872–1962).

9. Sakaguchi Test
This test is specific for arginine because this reaction is given by guanidinium compound.
The arginine reacts with α – napththol and an oxidizing agent  such as bromine water or sodium
hypochlorite/sodium hypobromite to give a red colored product. The other guanidinium
containing compounds other than amino acid also give this reaction.

Result interpretation:
 Positive sakaguchi test: Red color
 Negative sakaguchi test: no red color

10. Diacetyl Test

Voges and Proskauer, in 1898, first observed the production of a red color after the
addition of potassium hydroxide to cultures grown on specific media. Harden later
revealed that the development of the red color was a result of acetyl-methyl carbinol
production. In 1936 Barrit made the test more sensitive by adding alpha-naphthol to the
medium before adding potassium hydroxide.
The Voges-Proskauer (VP) test or Diacetyl Test is used to determine if an
organism produces acetylmethyl carbinol from glucose fermentation. If present,
acetylmethyl carbinol is converted to diacetyl in the presence of ∝- naphthol, strong
alkali (40% KOH), and atmospheric oxygen. The ∝-naphthol was not part of the original
procedure but was found to act as a color intensifier by Barritt and must be added first.
The diacetyl and quanidine-containing compounds found in the peptones of the broth then
condense to form a pinkish red polymer with a green fluorescence. This reaction is obtained
in the presence of arginine.

Positive Reaction: A pink-red color at the surface

Examples: Viridans group streptococci (except Streptococcus vestibularis),
Listeria, Enterobacter, Klebsiella, Serratia marcescens, Hafnia
alvei, Vibrio eltor, Vibrio alginolyticus, etc.

4 | NOTRE DAME OF MARBEL UNIVERSITY

City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

Negative Reaction: A lack of a pink-red color

Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella,
Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and
Vibrio parahaemolyticus etc.
A copper color should be considered negative. A rust color is a weak
positive  reaction

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5 | NOTRE DAME OF MARBEL UNIVERSITY

City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

11. Nitroprusside Reaction

Proteins containing cysteine give a reddish color with sodium nitroprusside in
dilute ammoniacal solution. This reaction is due to the presence of the SH group.
12. Bromine-Water Reaction
Tryptophan gives a pink color when treated with bromine water.

II. LEARNING OUTCOMES:

A. Distinguish amino acids from other organic compounds through the specific and general color
tests.
B. Describe the different chemicals involved for each test reactions and give the color each tests.
C. Perform the following tests in the laboratory and show the actual results.
D. Summarize and formulate a table of the different color tests, according to its principles, chemicals
used and the positive result of each of the tests.

III. REQUIREMENTS:
A. Materials: 24 test tubes, 10mL graduated cylinder, Alcohol lamp, electric water bath, wash
bottle, 2 test tube racks, pipettes

B. Chemicals: 0.1% CuSO4, Egg albumin solution, Biuret’s reagent, Hopkins – cole reagent,
Million’s reagents, 10% NaOH solution, Casein solution, HNO3 Concentrated, 1% Lead Acetate, 6M
NaOH, Glycine, Proline, Arginine, Other Amino acids, Xanthoproteic acid test reagents, Conc.
H2SO4, Albumin solution, Gelatin solution, 1% alpha-naphthol in alcohol, 2% Ninhydrin solution in
acetone, ethanol, Sodium hypochlorite solution, 1% Urea solution.

IV. PROCEDURES
1. Biuret’s Test
a. Thoroughly mix 2mL of 10% NaOH with 1 mL of the test solutions.
b. Add 1 drop of 0.1% CuSO4 solution.
c. Mix thoroughly and note if a pink or violet coloration develops. If not, add additional drops
(up to 10) of 0.1% CuSO4, mixing the solution after addition.
d. A positive test is the appearance of a pink or violet coloration; it may be very pale. Use a
white background.

2. Millon’s Test
a. Add 4 drops of million’s reagent to 1mL of the tests solutions.
b. Mix well, and observe carefully whether a white precipitate forms.
c. Warm the solution carefully by putting in water bath until the first appearance of a red color.
This
result constitutes a positive test.

3. Xanthoproteic Test
a. Add 1mL of concentrated nitric acid to 1 mL of all test solutions . Mix and note the
appearance of any heavy white precipitate.
b. Warm the mixture carefully, nothing any change to a yellow colored solution.

6 | NOTRE DAME OF MARBEL UNIVERSITY

City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

c. Cool the mixture in a stream of cold water and carefully add conc. HNO 3 and note if
the
color deepens to orange. This is a positive test.

4. Hopkin’s – Cole Test

a. Add 1 mL of Hopkins – Cole reagent to 1mL of the test solutions in a test tube.
b. Incline the tube at an angle and carefully add 1mL of concentrated H2SO4 so that it drawn
down one side and forms a separate lower layer in the tube.
c. A positive test consists of the appearance of a violet ring interface in a few seconds. If
this
fails to appears, very gently agitate the tube to provide slight mixing at the interface
without bringing about extensive solution of the layers in each other.

5. Reduce Sulfur Test

a. To 1mL of the test solutions, add 1 mL of 6M NaOH, and heat it carefully in a flame for
15
seconds (since protein solutions foam badly, be ready to move the tube out of the
flame
quickly).
b. Add 5 drops of lead acetate solution, if a black precipitate of lead sulfide does not form,
try
boiling the mixture again. (This precipitate maybe so finely dispersed it looks brown).

6. Sakaguchi Test
a. To 1mL of the test solutions, add 2 drops of alpha-naphthol solution and mix well.
b. Now add 2 mL of sodium hypochlorite solution.
c. Immediately add 1 ml of the 1% urea solution to establish if a red complex is formed.

7. Ninhydrin Test
a. To 1mL of the test solutions, add to each test solutions 5 drops of 2% Ninhydrin solution
b. Place the mixtures to a warm bath for approximately 5 minutes.
c. The development of a blue/violet colour indicates the presence of amino acids.

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City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

Activity No. 6 Report Sheet

COLOR REACTION OF PROTEINS
Name: _________________________ Rating: _________________
Date Performed: _________________ Date Submitted: _______________

I. RESULTS AND OBSERVATIONS

I. A. Describe the results of each Colour Tests and indicate whether the samples given are positive or
negative in the test samples.

Color Tests Inference of each Albumin Casein Gelatin

color tests

Biuret

Million’s

Xanthoproteic

Hopkin’s –
Cole

Reduced
Sulfur

Sakaguchi
Test

Ninhydrin

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City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

Test

Color Tests Amino Amino Amino Amino Amino

Acid_______ Acid_______ Acid_______ Acid_______ Acid_______

Biuret

Million’s

Xanthoprotei
c

Hopkin’s -
Cole

Reduced
Sulfur

Sakaguchi
Test

Ninhydrin
Test

II. GUIDE QUESTIONS:

1. Why does nitric acid stain the human skin yellow? Do all amino acids give a positive Xanthoproteic
test? Explain.

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City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

2. Which amino acids contain the benzene ring? What is the only acid that contains a phenolic group?
What is the only amino acid that contains the indole group?

3. According to advertisements, a commercial hair dressing contains “protein”. Which of the following
would be the best test to perform on the product to check this claim?

4. Will a dipeptide give a positive result in Biuret Test or not? Explain.

5. Indicate whether the following substances would give a positive (+) or negative (-) Biuret Test and
give a reason for your choice for every substances given below:

a. Tyrosylphenylalanin

b. Insulin

c. Glycylcysteinyltryptophan

d. Alanylglycine

6. Why would the Ninhydrin Test be more useful and reliable for showing the absence rather than the
presence of proteins? Give the uses of Ninhydrin Test.

10 | NOTRE DAME OF MARBEL UNIVERSITY

City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

III. CONCLUSION

IV. REFERENCES

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City of Koronadal, South Cotabato
Ana Liza D. Polido, MS
Laboratory Manual in Biochemistry
(MC 102L/BIOTC 104)

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City of Koronadal, South Cotabato