PROTEIN DENATURATION

Abstract:
Denaturation of proteins has an important role in food processing operations. It can range
from lowering of milk pH to form cottage cheese to heating beaten egg white to form meringues.
The objective of the lab session was to study effects of different factors on milk and egg proteins.
Albumin and casein were subjected to heat, pH changes, ethanol, lead nitrate, silver nitrate and
tannic acid. The effects of these agents on proteins were noted.
Introduction:
Denaturation of proteins changes their secondary, tertiary or quaternary structure.
Factors, that cause protein denaturation, are usually mild and therefore do not change the primary
structure of proteins by breaking peptide bonds.
In order to study protein denaturation it is, therefore, important to have an understanding
of different levels of protein structure. The sequence in which amino acids are linked is called the
primary structure. The three dimensional configuration of these chain segments, resulting from
hydrogen bonding between amino acids separated from each other along the chain, is called the
secondary structure. The two forms are alpha-helix and pleated-sheets. Tertiary structure is the
three dimensional shape of the whole polypeptide chain. This is due to the interaction between
amino acid side chains by hydrogen bonding, disulfide bonding, salt bridges (ionic interactions)
and hydrophobic interactions. Proteins having more than one chain form quaternary structures.
Factors such as heat, pH change, organic solvents or heavy metal ions may impact the aforesaid
interactions responsible for the three dimensional structure of the protein and as a result the
protein may be disrupted and loose its biological activity and is said to be denatured. This may
result in coagulation and precipitation of proteins from solution.
It is important to learn the mechanism by which individual denaturing agents work. Heat
causes kinetic energy increase which breaks hydrogen bonds or hydrophobic interactions. Salt
bridges (ionic interactions) between acid and basic functional groups of aminoacids can be
disrupted by changes in pH. Also pH change can cause protein precipitation by virtue of its
isoelectric point i.e., when the molecules are neutral and can approach each other and precipitate
out of solutions. Ethanol is capable of forming hydrogen bonds with protein and resultantly
disrupting the intermolecular hydrogen bonds within the molecule. Metal salts of mercury, lead
and silver can form bonds with disulfide groups and carboxylic groups thereby resulting in an
insoluble metal protein salt. Finally negative ions of tannic acid combine with basic positively
charged aminoacids to form a precipitate.
In the laboratory this phenomenon was studied on casein and albumin. Casein, a
phosphoprotein, found in milk as calcium salt exists as a soluble complex comprising of , , and

 caseins. , and casein are insoluble singly. Moreover, calcium caseinate has isoelectric point
at pH 4.6 and pH lowering precipitates it.
Albumin is a globular protein that is soluble in water and dilute salt solutions but
coagulated by heat. At a temperature of 70C it is irreversibly coagulated. Coagulated albumin is
insoluble in water. Exposure to ethanol also coagulates albumin and strong acids also coagulate
it. Albumin and casein form a precipitate by the addition of salts of metals.

Materials and Methods

To study the effect of heat on albumin, a roughly 2ml solution of 2% albumin (in-house)
was placed in a beaker containing water on a hot water plate and heated. To check effect of pH
again 2ml of 2% albumin and 2% casein (in-house) solutions were subjected to drop wise
introduction of sodium hydroxide till pH change to 14. 0.5% sodium bicarbonate was added to
next set of test tubes till pH change to 9 and finally hydrochloric acid was introduced to another
set of albumin and casein test tubes till pH change of 2.
Similarly albumin and casein solutions were taken in several test tubes and 95% ethanol,
lead nitrate, silver nitrate and tannic acid were added to labeled test tubes. Control solutions were
also maintained to compare the changes observed in the test solutions.
Results and discussion:
Results of the effect of various denaturing agents are given in table below:
Denaturing Procedure
Control
Heat (using hot plate)
pH : 10% Sodium Hydroxide

pH: 0.5% Sodium Bicarbonate

pH: 2% Hydrochloric Acid

Ethanol

2% Albumin
2% Casein
Light white color
Slightly Cloudy
Became opaque white as
compared to control solution.
Light white color
The solution became slightly
clearer after addition of 10%
sodium hydroxide.
Light white color
Almost no change from
control.
Turbid
Coagulation
was
seen
immediately after addition of
but disappeared soon and the
solution
became
slightly
clearer as compared to control.
Slight color change and more Solution became clearer and
opaque
no visible change seen.

Lead Nitrate

More cloudier than control

Heavy white coagulation

which settled to the bottom of
the test tube.
Silver Nitrate
Coagulation and precipitation Coagulation
of
protein
observed.
Tannic Acid
Cloudier and slight color Color of solution became
change
slightly buff and solution
became relatively clear.
Table 1: Effects of denaturing agents on 2% albumin and casein solutions
Albumin is profoundly affected by heat and irreversibly coagulated. Casein dissolves in
basic solution. It forms sodium caseinate with sodium hydroxide. This has an important
application in food industry to increase protein content of food products. When hydrochloric acid
is slowly added to the solution, precipitation or coagulation was seen as an isoelectric precipitate
is formed, but further addition of acid dissolved the precipitates possibly due to acid hydrolysis.
These observations support the effects of these denaturing agents mentioned in the text above.
References:
1. Vickie Vaclavik, Elizabeth W. Christian, Proteins in foods, In: Dennis R. Heldman, editor.
Essentials of Food Science. Springer Science
2. Zoubida Akkouche, Lyes Aissat, Khodia Madani, Effect of Heat on Egg White Proteins,
international Conference on Applied Life Sciences, 2012
3. Vincent Dormer Harris, Hand book of physiology, Page 106
4. Iraj Mehrnia, 2016, Foreign Materials in Food Products, Iraj Mehrnia, Food Processing-I
Laboratory Procedures, September 2016.
Questions:
1. Which method appeared to have the most dramatic denaturing affect on egg
albumin? Why do you think this method had a greater affect?
Heat had the most visibly profound effect on denaturation of egg albumin. When
subjected to heat, the globular structure of albumin is subject to denaturation. Depending
on the duration and intensity of heat, the denaturation can range from gelation to
coagulation. Various studies on heat coagulation of egg albumin mention a irreversible
coagulation upon heating. The first step in this coagulation is formation of disulfide
bonds and exposure of hydrophobic groups. Further heating causes polymerization by
intermolecular sulfhydryl-disulfide exchange.
2. Of the methods you tested, which would be more likely to be used in food industry?

Precipitation of milk casein by lowering pH has an important application in food industry.

This principle is used in curdling of milk. A small amount of curd containing
fermentation product of lactose in form of lactic acid is added to milk thereby bringing its
pH to isoelectric point of casein at which stage it precipitates forming white curds also
known as cottage cheese.