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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. ## Back to the list of abstracts