Thermodynamic Characterization of the Partially Unfolded State of Ca~(2+)-Loaded Bovine α-Lactalbumin: Evidence That Partial Unfolding Can Precede Ca~(2+) Release

摘要

The thermal denaturation of boyine α-lactalbumin (BLA) was studied at pH 7.5 and at various. Ca~(2+) concentrations using near-UV circular uichioism and differential scanning calorimetry. The Ca~(2+) dependence of the denaturation equilibria provesthat, in the transition region, partially unfolded a-lactalbumin consists of a mixture of Ca~(2+)-loaded and Ca~(2+)-free protein The thermodynamic parameters of the unfolding of these two species were determined at 68°C and were then compared with oneother, with the thermodynamic parameters deduced from calorimetric titration of a-lactalbumin with Ca~(2+) and with those derived from Ca~(2+) titration of a mutant human lysozyme having an engineered Ca~(2+)-binding site. This comparison indicated that(a) the unfolding curves for Ca~(2+)-BLA deduced from the near-UV ellipticity change are mere able to distinguish between unfolding with and without Ca~(2+) release than those deduced from differential scanning carorimetry, (b) the Ca~(2+)-loaded denaturated state of BLA is more folded than the Ca~(2+)-free protein at 68°C, and (c) a heat-induced unfolding process, consisting of an initial Ca~(2+) release, followed by a conformational relaxation, is unlikely to occur at the experimental pH and in themillimolar region of Ca~(2+) concentrations, due to the large free energy requirement of the initial step. A more probable mechanism would be unfolding via a Ca~(2+)-loaded intermediately unfolded state, with subsequent Ca~(2+) release.