EXPERIMENT NO.

PROTEIN DENATURATION

DUMADAG, JOSE ANTONIO

EDRIS, ABDULLA

SINDATOC, AROUFH

BIRUAR, BAI ALMIRA

EBALLE, ROVELYN ANNE

LUCES, GIANNA

NAVALES, JOANNE

TABJAN, SHAINA MAE

BSRT- 1B

GROUP 2
 INTRODUCTION

Protein is an essential macronutrient. It is found throughout the body—in muscle,

bone, skin, hair, and virtually every other body part or tissue. It makes up the enzymes

that power many chemical reactions and the hemoglobin that carries oxygen in your

blood. At least 10,000 different proteins make you what you are and keep you that way.

Proteins are polymers of amino acids. A typical protein may be composed of

hundreds of amino acids. The R-groups of the amino acid may be nonpolar, polar,

positively charged, or negatively charged. The primary structure of a protein is the

sequence of amino acids, and the secondary and tertiary structures of proteins define

the proteins folded state.

Changing the conformation of a protein either temporarily or permanently by

disrupting these forces is called denaturation. Denaturation may result in coagulation

with the protein being precipitated from solution. Since the native conformation is

usually the most water soluble, disrupting the secondary and tertiary structures causes

changes in solubility and frequently results in the formation of a solid in the solution.

Reagents or conditions that can cause denaturation are called denaturing agents; these

include heat, pH changes, alcohol, and heavy metal salts.

 I. OBJECTIVE

At the end of the experiment, we, the students, should be able to:

 Observe several chemical properties of amino acids and proteins;

 Observe the effects of several denaturing agents on a protein sample;
 Extract proteins from blood plasma; and
 Identify some amino acids through their reactions from specific reagents.

II. PROCEDURES
a. Add 2.0mL of egg white solution to each of the two (2) test tubes labeled 1 and 2.
b. To test tube 1, add 1.0mL of 1% AgNO3 solution and to test tube 2, add 1.0mL of

10% Pb(CH3COO)2 solution. Mix well and note the color of the precipitates

formed. Set aside for 5 minutes.

c. Decant the supernatant liquid and test the solubility of a small portion of the

precipitate in 5.0mL of water.

III. PRESENTATION OF RESULTS

EFFECT OF HEAVY METALS

Color of Precipitate Solubility in Water

Silver Nitrate Dark Yellow Insoluble

Lead Acetate Black Soluble

IV. DISCUSSION/ INTERPRETATION OF RESULTS

Silver nitrate is what is known as an “acidic salt”. Water solutions of it will have a

pH less than 7. It is an acidic salt because the acid dissociation constant (Ka) of the

silver ion is significantly larger than the base dissociation constant of the nitrate ion. The
egg whites in the silver nitrate will denature the egg quickly because the silver will react

with the egg whites and water, oxidizing the silver and changing its color to yellow.

Proteins in things like eggs possess a delicate structure or folding pattern.

Proteins are denatured by acidic solutions because H+ ions (also OH- ions or heat) can

break the hydrogen bonds that maintain the original structure of the proteins. After the

hydrogen bonds are broken the protein structure will change shape and new hydrogen

bonds will form that often result in a more rigid/firm structure. H+ ions can also catalyze

the hydrolysis of the peptide bonds in the proteins (egg albumin). Heavy metals may

also disrupt disulfide bonds because of their high affinity and attraction for sulfur and will

also lead to the denaturation of proteins.

For lead acetate and egg white solution, the product of this reaction is black lead

(II) sulfide. It forms upon the reaction between lead (II) acetate and the sulfur in the egg.

Because of their peptide structure and the presence of different amino acid groups in

their molecules proteins react with a variety of agents to form colored products.

PROTEIN DENATURATION

1. Define denaturation.
 A process in which the folding structure of a protein is altered due to exposure to

certain chemical or physical factors (e.g. heat, acid, solvents, etc.), causing the protein

to become biologically inactive.

2. What physical and chemical agents are capable of denaturing proteins? Give the

type of bonds or attractive interactions disrupted by these denaturing agents.

There 6 capable ways of denaturing the protein, these are the temperatures or heat,

the pH level, organic solvents, detergents, heavy metals, and mechanical stress. These

six (6) are the ways or reason that will denature the proteins. For the temperature; as

the temperature increases the molecular movement within the protein begins to vibrate

violently. A pH change to more acidic or more basic conditions can induce unfolding.

Acid-induced unfolding often occurs between pH 2 and 5, base-induced unfolding

usually requires pH 10 or higher. For the organic solvents, it disrupts the hydrogen

bonds within the protein. In the detergents, it affects or disrupts the hydrophobic and

hydrophilic region where it causes the protein chain to unfold. For the heavy metals, it

contains high atomic weights. For example, the salt of heavy metals, since it is ionic

they disrupt the salt bridges in proteins. In which it results to the insoluble protein salt or

even its biological activity. The last one is the mechanical stress, when you apply stress

like stirring or whipping the bonds that keep the 3D structures together, it weaken or

break down altogether, causing the protein to unfold and lose its properties.

3. What concentration of alcohol is most effective as a disinfectant? Why?

 The most effective concentration of alcohol is 70% Isopropyl alcohol or IPA,

particularly in solutions between 60% and 90% alcohol and 10 – 40% purified water, is

rapidly antimicrobial against bacteria, fungi, and viruses. 70% IPA solutions penetrate

the cell wall more completely which permeates the entire cell, coagulates all proteins,

and therefore the microorganism dies.

4. Explain how protein denaturation using heat, alcohol, and heavy metal ions is

used in the medical field.

Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic

interactions. This occurs because it increases the kinetic energy and causes the

molecules to vibrate rapidly and violently causing the bonds to disrupt. Medical supplies

and instruments are sterilized by heating to denature proteins in bacteria and thus

destroy the bacteria. A 70% alcohol solution is used as a disinfectant on the skin. This

concentration of alcohol is able to penetrate the bacterial cell wall and denature the

proteins and enzymes inside of the cell. A 95% alcohol solution merely coagulates the

protein on the outside of the cell wall and prevents any alcohol from entering the cell.

Alcohol denatures proteins by disrupting the side chain intermolecular hydrogen

bonding. Heavy metals may also disrupt disulfide bonds because of their high affinity

and attraction for sulfur and will also lead to the denaturation of proteins.

5. What amino acids in a protein are reactive with heavy metal ions? Explain and

illustrate with an equation.

Most of the amino acid in protein with carboxyl groups of acidic amino acids were can

be reactive to heavy metal ions. These are the amino acids that have a side chain at a

neutral pH because their side chain has an ionized carboxylic acid group whose pKa’s

are low enough to lose protons that why it becomes negatively charge in the process to

be acidic. Furthermore, electronegative substituents amino acids near the carboxyl

group act to increase the acidity. Example, heavy metal ions like (Pb2+, Ag+, Hg2+ that

can react with -COO; or OH group of amino acid. Their electronic and molecular

structures, cohesive energies, and stiffness of the local potential energy well at the

cation (M) site are determined and attempts are made to understand the diversity in

geometry and the properties of binding of different metal ions with -COOH group.

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