THERMODYNAMIC CHARACTERIZATION OF THE PARTIALLY UNFOLDED STATE OF CA2-LOADED BOVINE ALPHA-LACTALBUMIN - EVIDENCE THAT PARTIAL UNFOLDING CANPRECEDE CA2+ RELEASE()

The thermal denaturation of bovine alpha-lactalbumin (BLA) was studied at pH 7.5 and at various Ca2+ concentrations using near-UV circular d ichroism and differential scanning calorimetry. The Ca2+ dependence of the denaturation equilibria proves that, in the transition region, pa rtially unfolded alpha-lactalbumin consists of a mixture of Ca2+-loade d and Ca2+-free protein. The thermodynamic parameters of the unfolding of these two species were determined at 68 degrees C and were then co mpared with one other, with the thermodynamic parameters deduced from calorimetric titration of alpha-lactalbumin with Ca2+, and with those derived from Ca2+ titration of a mutant human lysozyme having an engin eered Ca2+-binding site. This comparison indicated that (a) the unfold ing curves for Ca2+-BLA deduced from the near-UV ellipticity change ar e more able to distinguish between unfolding with and without Ca2+ rel ease than those deduced from differential scanning calorimetry, (b) th e Ca2+-loaded denaturated state of BLA is more folded than the Ca2+-fr ee protein at 68 degrees C, and (c) a heat-induced unfolding process, consisting of an initial Ca2+ release, followed by a conformational re laxation, is unlikely to occur at the experimental pH and in the milli molar region of Ca2+ concentrations, due to the large free energy requ irement of the initial step. A more probable mechanism would be unfold ing via a Ca2+-loaded intermediately unfolded slate, with subsequent C a2+ release.