ACTIVITY 2.

PROTEIN DENATURATION

GROUP #: ________ LEADER______________Date: _____________________

Name _____________________ Year & Section: ____________

I. OBJECTIVES

At the end of this experiment, you will be able to:

a. explain what protein denaturation is

b. compare and examine the different reactions involved in the denaturation of
proteins

II. INTRODUCTION

Denaturation is the loss of the secondary, tertiary, and quaternary structures of a

protein by a chemical or physical agent that leaves the primary structure intact.  

The disruption of the primary protein structure occurs with the hydrolysis of the
peptide bonds to produce free amino acids. 

If these changes occur to a small extent, denaturation can be reversed.  For

example, a denatured protein can be removed from a urea solution and put it back
into the water, it often reassumes its secondary and tertiary structures.  This
process is called reversible denaturation. 

 In living cells, some denaturation caused by heat can be reversed by chaperones. 
These proteins help a partially heat-denatured protein to regain its native
secondary, tertiary, and quaternary structures. Some denaturation, however, is
irreversible.

Factors that Cause Protein Denaturation:

1. Strong acids and bases

Changes in the pH can disrupt hydrogen bonds and salt bridges, causing irreversible
denaturation.  Proteins are coagulated by strong acids like concentrated HCl,
H2SO4, and HNO3.  Alkaloidal reagents such as tannic acid and picric acid form
insoluble compounds with protein.  It denatures protein irreversibly by disrupting
the salt bridges and the disulfide bonds.

2. Organic Solvents

Alcohol coagulates all types of protein except prolamines.  Alcohol irreversibly

denatures by forming hydrogen bonds that compete with the naturally occurring
hydrogen bonds in the protein.  At a concentration of 70%, ethanol penetrates
bacteria and kills them by coagulating their proteins, whereas 95% of alcohol
denatures only surface proteins.

3. Reducing Agents

Reducing agents, such as 2-mercaptoethanol (HOCH2CH2SH), can break the –S-S-

disulfide bonds, reducing them to -SH groups.  Reducing agents also disrupts
hydrogen bonds and hydrophobic interaction.

4. Salt Concentration

Changes in the salt concentration may result in the formation of a precipitate

(salting out).  Salts affect both salt bridges and hydrogen bonds.

5. Heavy Metals

Heavy metal salts like mercuric chloride or silver nitrate, and lead precipitate
protein.  It also cleaves –SH bonds.  It denatures protein irreversibly by disrupting
the salt bridges and the disulfide bonds.

6. Temperature Change

Heat cleaves hydrogen bonds, so boiling a protein solution destroys the α-helical
and β-pleated sheet structure.

7. Mechanical Stress

Stirring and grinding action may disrupt the delicate balance forces required to
maintain protein structure.
III. MATERIALS

         2 eggs                                             small pan

         Bleach                                            fork or whisk

         Rubbing Alcohol                  glasses or container

IV. PROCEDURE

1. Separate the yolks from the egg white.

2. Split the egg white into five equal parts and place each in different
containers.
3. Add an equal amount of water to the 1st portion (cup A). This will serve
as your control.
4. Add an equal amount of bleach to the 2nd portion (cup B). Wait for 30
minutes.
5. Add an equal amount of alcohol to the 3rd portion (cup C). Wait for 30
minutes.
6. Put the 4th portion (cup D) in a small pan and heat it over low flame (or
pop it in the microwave for 5 minutes). BE CAREFUL.
7. Beat the 5th portion using a fork or whisk for 10 minutes.

What happened to each egg set up?

Record your observation and prepare your report. You can submit a written report,
or PowerPoint presentation.