THE
SCIENTIFIC EXPLANATION
Thermal
Denaturation of Natural Milk Proteins
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When proteins
are exposed to increasing temperature, losses of solubility
or enzymatic activity occurs over a fairly narrow
range. Depending upon the protein studied and the
severity of the heating, these changes may or may
not be reversible.
As
the temperature is increased, a number of bonds in
the protein molecule are weakened. The first affected
are the long range interactions that are necessary
for the presence of tertiary structure. As these bonds
are first weakened and are broken, the protein obtains
a more flexible structure and the groups are exposed
to solvent. If heating ceases at this stage the protein
should be able to readily refold to the native structure.
As
heating continues, some of the cooperative hydrogen
bonds that stabilize helical structure will begin
to break. As these bonds are broken, water can interact
with and form new hydrogen bonds with the amide nitrogen
and carbonyl oxygens of the peptide bonds.
The
presence of water further weakens nearby hydrogen
bonds by causing an increase in the effective dielectric
constant near them. As the helical structure is broken,
hydrophobic groups are exposed to the solvent.
The
effect of exposure of new hydrogen bonding groups
and of hydrophobic groups is to increase the amount
of water bound by the protein molecules. The unfolding
that occurs increase the hydrodynamic radius of the
molecule causing the viscosity of the solution to
increase. The net result will be an attempt by the
protein to minimize its free energy by burying as
many hydrophobic groups while exposing as many polar
groups as possible to the solvent. While this is analogous
to what occurred when the protein folded originally,
it is happening at a much higher temperature. This
greatly weakens the short range interaction that initially
direct protein folding and the structures that occur
will often be vastly different from the native protein.
Upon
cooling, the structures obtained by the aggregated
proteins may not be those of lowest possible free
energy, but kinetic barriers will prevent them from
returning to the native format. Any attempt to obtain
the native structure would first require that the
hydrophobic bonds that caused the aggregation be broken.
This
would be energetically unfavorable and highly unlikely.
Only when all the intermolecular hydrophobic bonds
were broken, could the protein begin to refold as
directed by the energy of short range interactions.
The exposure of this large number of hydrophobic groups
to the solvent, however, presents a large energy barrier
that make such a refolding kinetically unlikely.
Exposure
of most proteins to high temperatures results in irreversible
denaturation. Some proteins, like caseins, however,
contain little if any secondary structure and have
managed to remove their hydrophobic groups from contact
with the solvent without the need for extensive structure.
This lack of secondary structure causes these proteins
to be extremely resistant to thermal denaturation.
The
increased water binding noted in the early stages
of denaturation may be retained following hydrophobic
aggregations. The loss of solubility that occurs will
greatly reduce the viscosity to a level below that
of the native proteins.
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