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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.
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