Chemistry 27A Formal Report

Experiment 5

STABILITY OF PROTEINS

Tue-Thu 4:00 7:00

Ofel Jireh T. Montenegro

BS Biology II

Carl Michael Y. Labtog

Lab Instructor

Tue-Thu 4:00 7:00

July 28, 2011

I- Introduction
When a solution of a protein is boiled, the protein frequently becomes insolublei.e., it
is denaturedand remains insoluble even when the solution is cooled. The denaturation of the
proteins of egg white by heatas when boiling an eggis an example of irreversible
denaturation. The denatured protein has the same primary structure as the original, or native,
protein. The weak forces between charged groups and the weaker forces of mutual attraction
of nonpolar groups are disrupted at elevated temperatures, however; as a result, the tertiary
structure of the protein is lost. In some instances the original structure of the protein can be
regenerated; the process is called renaturation.
Denaturation can be brought about in various ways. Proteins are denatured by
treatment with alkaline or acid, oxidizing or reducing agents, and certain organic solvents.
Interesting among denaturing agents are those that affect the secondary and tertiary structure
without affecting the primary structure. The agents most frequently used for this purpose are
urea and guanidinium chloride. These molecules, because of their high affinity for peptide
bonds, break the hydrogen bonds and the salt bridges between positive and negative side
chains, thereby abolishing the tertiary structure of the peptide chain. When denaturing agents
are removed from a protein solution, the native protein re-forms in many cases. Denaturation
can also be accomplished by reduction of the disulfide bonds of cystinei.e., conversion of the
disulfide bond (SS) to two sulfhydryl groups (SH). This, of course, results in the formation
of two cysteines. Reoxidation of the cysteines by exposure to air sometimes regenerates the
native protein. In other cases, however, the wrong cysteines become bound to each other,
resulting in a different protein. Finally, denaturation can also be accomplished by exposing
proteins to organic solvents such as ethanol or acetone. It is believed that the organic solvents
interfere with the mutual attraction of nonpolar groups.
Some of the smaller proteins, however, are extremely stable, even against heat; for
example, solutions of ribonuclease can be exposed for short periods of time to temperatures of
90 C (194 F) without undergoing significant denaturation. Denaturation does not involve
identical changes in protein molecules; a common property of denatured proteins, however, is
the loss of biological activitye.g., the ability to act as enzymes or hormones.
Although denaturation had long been considered an all-or-none reaction, it is now
thought that many intermediary states exist between native and denatured protein. In some
instances, however, the breaking of a key bond could be followed by the complete breakdown
of the conformation of the native protein.
Although many native proteins are resistant to the action of the enzyme trypsin, which
breaks down proteins during digestion, they are hydrolyzed by the same enzyme after

denaturation. Evidently, the peptide bonds that can be split by trypsin are inaccessible in the
native proteins but become accessible during denaturation. Similarly, denatured proteins give
more intense colour reactions for tyrosine, histidine, and arginine than do the same proteins in
the native state. The increased accessibility of reactive groups of denatured proteins is
attributed to an unfolding of the peptide chains.

II- Methodology
A. Procedure
Denaturation of Proteins
1. Three mL of neutral egg albumin solution was placed into four test tubes. One is
added with 5% acetic acid, to another 2 of 1% sodium carbonate solution, to the third was two
drops of 5% NaCl solution. The following test tubes were labeled.
2. The four test tubes were placed in small beaker of water with thermometer on the
fourth test tube. Then the samples were heated. Then the temperatures at which the
coagulation begins and ends in each test tube.
Isoelectric Precipitation
Each protein solutions (gelatin and albumin) was added 0.10 M HCl drop by drop until
the maximum precipitation occurs. Then the addition of HCl was continued, shaking the tube
after each addition. Then 0.10 N NaOH was added drop by drop. Then the observations were
recorded.

Denaturation by Concentrated Minerals Acids (HNO3, HCl, H2SO4)

Two mL of the samples as added with the following concentrated acids drop by drop,
and then it was mixed through shaking. Until an excess acid has been added, then it was
observed whether an excess of the acid will redissolve the precipitate.
Denaturation by Organic Solvents
Two mL of the samples was added with ethanol and acetic acid, drop by drop until
precipitate as obtained.

Denaturation by Salts of Heavy Metals

Two mL of the samples was added 2% lead acetate drop by drop until a precipitate is
obtained.
Denaturation by Alkaloidal Reagents
Few drops of saturated picric acid solution was added to 2mL of each sample. Then it
was observed.
Salting Out
Two mL each samples was added with 2mL saturate ammonium sulfate solution.

B. Materials and Apparatus

The chemicals used in the neutral egg albumin solution, 5% acetic acid, 1% sodium
carbonate solution, 5% NaCl, 0.10 M HCl, Gelatin, 0.10 N NaOH, HNO3, HCl, H2SO4, 2% lead
acetate,ethanol, picric acid solution, distilled water, and saturated ammonium sulfate.
The apparatus include test tubes, test tube brush, droppers, beakers, stirring rod,
thermometer, test tube holder and a source of heat.

III- Results:
DENATURATION BY HEAT
Solutions

Added
Reagent

After heating

Coagulated

Coagulation

Start

End

56

66

T1

5% Ch3COOH

T2

1% NaHCO3

Yellowish solution

86

T3

5% NaCl

Coagulated

65

77

T4

NONE

Cloudy solution

65

85

ISOELECTRIC PRECIPITATION
Protein solution
ALBUMIN

Reaction. w/ 0.10 M HCl

Reaction. w/ 0.10 M NaOH

white precipitate

GELATIN

white precipitate

white precipitate

cloudy solution

DENATURATION OF CONCENTRATED MINERAL ACIDS

Reagents: HNO3, HCl & H2SO4
Protein solution

Add. Of HNO3

ALBUMIN

White ppt.

GELATIN

clear yellowish
solution

Add. Of HCl
White ppt.
No ppt. formed

Add. Of H2SO4
Yellow soln
Brownish solution

DENATURATION OF ORGANIC SOLVENTS

Reagents: Ch3COOH and CH3CH2OH
Protein solution

Add. Of Ch3COOH

Add. Of CH3CH2OH

ALBUMIN

White ppt.

White ppt.

GELATIN

White ppt.

White ppt.

DENATURATION BY SALTS OF HEAVY METALS

Reagent: Pb (CH3COO)2
Protein solution

Addition of Pb (CH3COO)2

ALBUMIN

White ppt. formed

GELATIN

DENATURATION OF ALKALOIDAL REAGENTS

Reagent: saturated picric acid
Protein solution

Reaction with Picric Acid

ALBUMIN

Yellow cloudy solution

GELATIN

Yellow clear solution

SALTING OUT
Reagent: Saturated NH4SO4
Protein solution

Observation

ALBUMIN

White ppt. formed

GELATIN

No ppt. formed

IV- Discussion
Denaturation in biology, process modifying the molecular structure of a protein.
Denaturation involves the breaking of many of the weak linkages, or bonds (e.g., hydrogen
bonds), within a protein molecule that are responsible for the highly ordered structure of the
protein in its natural (native) state. Denatured proteins have a looser, more random structure;
most are insoluble. Denaturation can be brought about in various wayse.g., by heating, by
treatment with alkali, acid, urea, or detergents, and by vigorous shaking.
In denaturation by heat, test tube one and three, the samples coagulated. While in test
tube two, a yellowish solution was observed. In test tube four its still a cloudy solution.
In isoelectric precipitation the samples being mixed with 0.10 m HCl and 0.10 N NaOH
produced white precipitate except of that of albumin that just produce a cloudy solution.
In denaturation by concentrated mineral acids, upon addition if the following acids on
albumin it produced white precipitate and a yellow solution for H2SO4. For gelatin it produced a
clear yellow solution, no precipitate for HCl, and produced a brownish solution with H2SO4.
In denaturation by organic solvents the protein solutions produced white precipitate
when added with ethanol and acetic acid.

In denaturation by salts of heavy metals albumin produced a white precipitate when

added with lead acetate.
In salting out, albumin solution and saturated ammonium sulfate produced a white
precipitate, while in gelatin it did not produced.

V- Conclusion
This experiment tackles more about denaturation of proteins. These proteins can be
denatured at several ways. It can be brought by heating, by treatment with alkali, acid, urea, or
detergents, and by vigorous shaking. Therefore these reactions are vital to life especially on
natural processes like in humans. Denaturation is essential, a very simple example of which is
cooking, and we cook or process foods before consumption. The color changes during cooking
correspond to structural changes taking place in the meat. These structural changes are due to
the effects of heat on collagen (connective tissue protein) and actin and myosin (myofibrillar
proteins). Through this experiment we are able to observe visible chemical reactions. And
because of this we are able to know the importance of the following chemicals in the reaction.

VI- References
Encyclopedia Britannica
Chemistry 27 BIOCHEMISTRY Laboratory Manual by: Maria Rosario B. Nuenay, et. al.