COLLEGE OF PHARMACY

ONLINE LEARNING ACTIVITY

PH-PHR 127 LABORATORY – AY 2020-2021

SUBJECT CODE PH-PHR 127 LABORATORY

SUBJECT TITLE PHARMACEUTICAL BIOCHEMISTRY
EXPERIMENT Denaturation of Protein
• To observe the physical change when proteins denature;
• To identify the factors that lead to protein denaturation;
LEARNING OBJECTIVES • To compare the different denaturing agents; and
• To recognize the significance of protein denaturation in biological
preparations
Ryan Joseph C. Tuzon, RPh
PREPARED BY
Instructor II
DATE PREPARED June 13, 2020

THEORY
Proteins have rather complex structures in which the long chains of amino acids assume characteristic
three-dimensional shapes called the tertiary structures of proteins. The conformations of the side chains
and the positions of any prosthetic groups are parts of the tertiary structure, as is the arrangement of helical
and pleated-sheet sections with respect to one another. The interactions between the side chains play an
important role in the folding of proteins. The folding pattern frequently brings residues that are separated in
the amino acid sequence into proximity in the tertiary structure of the native protein. For any native protein,
there is one, or at most a few, three-dimensional structures that function correctly. Therefore, the
composition and order of amino acids, and hence, the conformation of the proteins are critical to the
biological function of the protein.

The three-dimensional conformation of a protein is the result of the interplay of all the stabilizing forces.
These noncovalent stabilizing interactions, however, are weak, and it is not surprising that they can be
disrupted easily. When a substance or condition disrupts the three-dimensional features of a protein’s
structure, the protein is said to be denatured. Denaturation usually causes a protein to lose its biological
activity. In addition, it often makes the protein less soluble, leading to coagulation or precipitation.

Proteins can be denatured by heat, pH, and chemicals. An increase in temperature favors vibrations within
the molecule, and the energy of these vibrations can become great enough to disrupt the tertiary structure.
At either high or low extremes of pH, at least some of the changes on the protein are missing, and so the
electrostatic interactions that would normally stabilize the native active form of the protein are drastically
reduced. Likewise, this leads to denaturation. The binding of detergents, such as sodium dodecyl sulfate
(SDS), also denatures proteins. Detergents tend to disrupt hydrophobic interactions. If a detergent is
charged, it can also disrupt electrostatic interactions within the protein. Other reagents that can denature
proteins are urea and guanidine hydrochloride, which disrupt hydrophobic interactions and form stronger
hydrogen bonds with the protein than those within the protein itself, and β-mercaptoethanol, which reduce
disulfide bridges to form sulfhydryl groups.

Some types of denaturation can be reversed, while others are permanent. In the former, if experimental
conditions are properly chosen, the native conformation of the protein can be recovered when denaturants
are removed. Experiments such as this shows that the amino acid sequence of the protein contains all the
information necessary for the folding of the protein. In this exercise, you will compare several denaturing
agents and processes.

MATERIALS
• 1% casein • 1% HgCl2
• alcoholic gliadin solution • 10% sodium ferrocyanide
• 1% ZnSO4 • isopropyl alcohol
• 1% CuSO4
 COLLEGE OF PHARMACY
ONLINE LEARNING ACTIVITY
PH-PHR 127 LABORATORY – AY 2020-2021

PROCEDURE
For each of the following tests, place 2 mL of 1% casein, alcoholic gliadin solution (obtained from
Experiment No. 2), and egg white in separate 20-mL test tubes to serve as a standard protein samples.
Then, perform each of the following tests on an individual 2-mL samples of 1% casein, alcoholic gliadin
solution, and egg white and record your observations. Use (NP) for no precipitation and (P) for precipitation.

The Effect of Heat

1. Place a test solution in a boiling water bath for 5 minutes.
2. Compare the appearance of the tube with the unheated standard.

The Effect of Alcohol

1. Add 2 mL of isopropyl alcohol to a test sample
2. Mix well.
3. Compare to the standard.

Dispose the solution containing isopropyl alcohol in the sink.

The Effect of Metals

1. To separate test samples, add 2 mL of 1% ZnSO 4, 1% CuSO4, 1% HgCl2, and 10% sodium
ferrocyanide.

Solutions of mercuric chloride extremely poisonous. Wear appropriate personal protective

equipment, such as safety goggles, gloves, industrial mask, and laboratory gown. Avoid
contact with skin.

2. Mix well and record your observations.

Dispose the solutions containing ZnSO4, CuSO4, and sodium ferrocyanide in the sink.
Dispose the solution containing HgCl2 in the Metal Residues container.
 COLLEGE OF PHARMACY
ONLINE LEARNING ACTIVITY
PH-PHR 127 LABORATORY – AY 2020-2021

LEARNING ACTIVITY
INSTRUCTIONS. Accomplish/Answer the following in a short-bond paper (handwritten or otherwise, for as long as the font is clear enough).
Make sure to indicate your names and group number in your submission. This activity must be accomplished by group and submitted within
the meeting.

1. Theoretical Results. Fill up the following table with theoretical results, using the format specified in the procedure.
Alcoholic Gliadin
1% Casein Egg White
Solution
Heat
Isopropyl Alcohol
1% ZnSO4
1% CuSO4
1% HgCl2
10% Sodium Ferrocyanide

2. Guide Questions. Answer the following guide questions.

a. How does ZnSO4, CuSO4, and HgCl2 cause protein denaturation? Which specific amino acid/ functional group do they disrupt?
b. How does Na4[Fe(CN)6] cause protein denaturation? Which specific amino acid/ functional group does it disrupt?