The energetics of structural changes in the holo
and apo forms of α-lactalbumin and the transition between
their native and denatured states induced by binding Ca2+
and Na+ have been studied by differential scanning
and isothermal titration microcalorimetry and circular
dichroism spectroscopy under various solvent conditions.
Removal of Ca2+ from the protein enhances its
sensitivity to pH and ionic conditions due to noncompensated
negative charge–charge interactions at the cation
binding site, which significantly reduces its overall stability.
At neutral pH and low ionic strength, the native structure
of apo-α-lactalbumin is stable below 14 °C and
undergoes a conformational change to a native-like molten
globule intermediate at temperatures above 25 °C.
The denaturation of either holo- or apo-α-lactalbumin
is a highly cooperative process that is characterized by
an enthalpy of similar magnitude when calculated at the
same temperature. Measured by direct calorimetric titration,
the enthalpy of Ca2+-binding to apo-LA at pH
7.5 is −7.1 kJ mol−1 at 5.0 °C,
which is essentially invariant to protonation effects.
This small enthalpy effect infers that stabilization of
α-lactalbumin by Ca2+ is primarily an entropy
driven process, presumably arising from electrostatic interactions
and the hydration effect. In contrast to the binding of
calcium, the interaction of sodium with apo-LA does not
produce a noticeable heat effect and is characterized by
its ionic nature rather than specific binding to the metal-binding
site. Characterization of the conformational stability
and ligand binding energetics of α-lactalbumin as a
function of solvent conditions furnishes significant insight
regarding the molecular flexibility and regulatory mechanism
mediated by this protein.