BIOCHEMISTRY MANUAL

Prepared by Admer Rey C. Dablio, v2012

Revised by Mary-Ann A. Landiao, v2016

Experiment 3
Score:

Chemical Reactions of Amino Acids and

Protein Functional Group
Name: Joann H. Justiniane
Groupmates: Marla C. Basa
Ana Margarita L.
Baytion

Date Performed: July

2016
Instructors Signature:

12,

Objectives:

II

To detect the presence of proteins or peptides.

To describe the test and positive results.
Chemicals:

III

2% Glycine
1% Tyrosine
2% Proline
0.5% Ninhydrin-ethanol
2% Arginine
3M NaOH
Concentrated H2SO4
2% Casein

0.1 CuSO4
2% Gelatin
2% Albumin
Concentrated HNO3
2% Tryptophan
2mL Hopkins-Cole
2% Nitroprusside

Apparatus/Materials/Equipment:

18 Test tubes
50 mL beaker
100 mL beaker
Stirring rod
Watch glass
Wire Gauze

Water bath
Test tube rack
Test tube holder
Hot plate
Bunsen Burner
Tong

IV

Schematic Diagram of the Procedure:

Please write on a short bond paper.

Summary of Theory
Proteins came from the Greek word proteios which means first.
Protein is a class of organic compound which is present in and important to
living organisms. It holds together, protect, and provide structure to the body
of a multi-celled organisms in the form of skin, hair, callus, cartilage, muscles,
tendons and ligaments. They catalyze, regulate, and protect the body
chemistry in the form of enzymes, hormones, antibodies, and globulins. In the
form of hemoglobin, myoglobin and various lipoproteins, they affect the
transport of oxygen and other substances within an organism.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

Each protein has its own precise function. The shapes of proteins are
important and these are determined by the sequence of amino acids that
made them up.
Amino acids are the building blocks of proteins. Twenty different amino
acids are used to make the body's proteins. Of these nine are
called essential meaning they can only be obtained from the food we eat) and
eleven are non-essential meaning they may be synthesized in the body
though they are usually obtained from food.
Proteins may be identified by three types of reactions:
a. Reactions which are due to the colloidal nature of the proteins. Example in
heat coagulation, salting out;
b. Reactions which are due to chemical reactions between the protein
molecule and the reagent and usually involve the acidity and basicity of
the protein molecule. Example is precipitation by a strong acids, salts, of
heavy metals or alkaloidal reagents.
c. Reaction which are due to the presence of specific chemical groups in the
protein molecule.
Certain functional groups in 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.
VI

Observations
A. Ninhydrin Test
Table A.1 Ninhydrin Test
Addition of 0.5%
Ninhydrinethanol
2% Glycine
Red Orange
1% Tyrosine
Brown
2% Proline
Red Orange
2% Casein
Red Orange
2% Gelatin
Red Orange

After Heating

Odor

Orange
Red
Red Orange
Light Orange
Red Orange

Iron
No odor
No odor
Urine
No odor

After heating

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

B. Biuret Test
Table B.1 Biuret Test
Before Addition of
CuSO4
2% Glycine
Colorless
2% Gelatin
Colorless
2% Casein
Yellow
2% Arginine
Colorless

After Addition of CuSO4

Clear Blue
Violet
Dark Green
Blurry Light Blue

After Shaking

C. Xanthoproteic Test
Table C.1 Xanthoproteic Test
Original
Color

Addition of
HNO3

After

Addition of
NaOH

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

1% Tyrosine

Brownish

Brown ppt.

Brown
precipitate

Yellowish

No change

2%
Tryptophan
2% Glycine

Colorless

No change

2% Gelatin

Colorless

No change

Slightly
immiscible
Slightly
immiscible
Colorless

After Heating

1
2
3
4
5

Dark yellow
solution and the
precipitate was
dissolved
Light yellow
No change
Yellow

After Addition of 3M NaOH

D.

D. Millons Test
Table D.1 Millons Test
Original
After Addition of
After Heating
Color
Millons reagent
Heating
1% Tyrosine After
Brown
Brown soln with ppt. Clear soln with dark brown ppt.
2% Glycine
Colorless
No change
No change
2%
Light yellow
Blurry yellow soln
Lighter yellow with yellow ppt.
Tryptophan
with yellow ppt.
2% Gelatin
Colorless
Slightly immiscible
Slightly immiscible
2% Casein
Yellow
Clear yellow
No changes in soln but there
was formation of small yellow
ppt.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

VII

Analysis
Proteins are essentially natural polymers composed of long chains of
subunits. These smaller units are called amino acids. One "end" of an amino
acid has acidic character because it has a carboxyl (COOH) functional group.
The other end has basic character because it has an amino (NH 2) functional
group. When two amino acids react, they form what is called a peptide bond.
The resulting molecule, called a dipeptide, still has one end that is acidic and
another that is basic. With this fundamental reactive pattern, it is possible to
string together many amino acids to form a polypeptide.
A. Ninhydrin Test
Common alpha amino acids, except proline undergo a unique reaction
with triketohydrindene hydrate known as Ninhydrin. The ninhydrin reagent
reacts with the amine functional group of -amino acids to form purplecolored compounds. Ninhydrin is used to detect fingerprints because it reacts
with amino acids from the proteins in skin cells transferred to the surface by
the individual leaving the fingerprint.

Amino acids that have secondary amino group attachments also react
with ninhydrin. However, when the amino group is secondary, the
condensation product is yellow. Blue-purple and yellow reaction products
positively identify free amino groups on amino acids and proteins.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

However in Table A.1, all of our solutions are orange or red orange.
The color varies slightly from acid to acid, probably because unreacted acids
complex with the pigment. Since all that is required for color development is
ammonia and partially reduced ninhydrin, the ammonium salts of weak and
strong acids, as well as certain amines, can
give a false positive result to the ninhydrin test. The striking color change is
due to the large change in electron confinement on formation of the anion.
B. Biuret Test
This test is specific for the peptide bond. Substances, containing not
less than two peptide linkages give positive result with this test. In this
reaction, proteins form a purple colored complex with CuSO 4 in a strongly
alkaline solution. A positive test is indicated by the formation of a violet color.
However in Table B.1, only the 2% gelatin gave a positive result when
it was added by CuSO4.

C. Xanthoproteic Test
Aromatic amino acids, such as Phenyl alanine, tyrosine and tryptophan,
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.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

The test gave a positive result in tyrosine, tryptophan and gelatin.

These proteins was carrying aromatic groups. Aromatic groups on these
compounds was nitrated by HNO3. The nitro derivate show an intensely yellow
color.
D. Millons Test
Millons test was developed by Auguste Millon, a French Chemist.
Phenolic amino acids such as Tyrosine and its derivatives respond to
this test. Compounds with a hydroxyl benzene radical react with Millons
reagent to form a red colored complex. Millons reagent is a solution of
mercuric sulphate in sulphuric acid.
In the four compounds used in Table D.1, only tyrosine and casein
have phenol functional group in their structure, but these compounds didnt
gave a positive result because it was heated well.

VIII

Conclusion
Therefore, Proteins can interact with one another and with other
biological macromolecules to form complex molecules. Some proteins are
quite rigid, whereas others display limited flexibility. The functions and
reactions of proteins are dependent on their structures. The building blocks of
protein called amino acids can both act as acid and base.
Some of the tests was not successful due to some personal error. The
experimenter failed to carry out some of tests because the reagents was not
available.
For further experimentation of the chemical reactions of amino acids
and protein functional groups, the experimenter recommends to study the
procedure well and prepare beforehand the reagents and apparatuses.

IX

References
1. Berg JM, Tymoczko JL, Stryer L., Biochemistry 5 th Edition, New York: W H
Freeman; 2002.
2. William R., Proteins, Peptides & Amino Acids, May 5, 2013
3. http://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_fu
nctional_group_in_protein.pdf (taken July 17, 2016)
4. Dr. Ian Hunt, Amino Acids, Peptides and Proteins.
5. vlab.amrita.edu, (2011). Qualitative Analysis of Amino Acid. Retrieved 17
July 2016, from vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
6. Sundin, Protein and Amino Acids, Organic Chemistry 3510.
7. Ball D. W., Hill J. W., Scott R. J., Introduction to Chemistry, General, organic,
and Biological, Vol. 1.0.
8. Senese F., What is a simple test for the presence of amino acids? 19972010.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY MANUAL
Prepared by Admer Rey C. Dablio, v2012
Revised by Mary-Ann A. Landiao, v2016

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.