Thermodynamic characterization of the partially unfolded state of
Ca2+-loaded bovine alpha-lactalbumin: evidence that partial unfolding
can precede Ca2+ release.

Geertrui Vanderheeren, Katholieke Universiteit Leuven campus Kortrijk
(KULAK) E. Sabbelaan 53 B-8500 Kortrijk Belgium

The thermal denaturation of bovine alpha-lactalbumin (BLA) was studied
at pH 7.5 and at various Ca2+ concentrations using near-UV circular
dichroism and differential scanning calorimetry. The Ca2+ dependence
of the denaturation equilibria proves that, in the transition region,
partially unfolded BLA consists of a mixture of Ca2+-loaded and
Ca2+-free protein.

The thermodynamic parameters of the unfolding of
these two species were determined at 680C and were then compared with
one another, with the thermodynamic parameters deduced from
calorimetric titration of BLA with Ca2+ and with those derived from
Ca2+ titration of a mutant human lysozyme having an engineered
Ca2+-binding site. This comparison indicated that (a) the unfolding
curves for Ca2+-BLA deduced from the near-UV ellipticity change are
more able to distinguish between unfolding with and without Ca2+
release than those deduced from differential scanning calorimetry, (b)
the Ca2+-loaded denatuated state of BLA is more folded than the
Ca2+-free protein at 680C, (c) a heat induced unfolding process,
consisting of an initial Ca2+ release, followed by a conformational
relaxation, is unlikely to occur at the experimental pH and in the mM
region of Ca2+ concentrations, due to the large free energy
requirement of the initial step. A more probable mechanism would be
unfolding via a Ca2+ loaded intermediately unfolded state, with
subsequent Ca2+ release.



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