Thermal unfolding of bovine alpha-lactalbumin. Comparison of circular dichroism with hydrophobicity measurements.

The thermal unfolding of apo- and Ca(2+)-loaded bovine alpha-lactalbumin (BLA) determined by circular dichroism measurements at 220 and 270 nm is related to the exposure of its hydrophobic surface measured by the interaction with 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonate. The results indicate that several (about 5) probe molecules are able to bind with low affinity to the compact surface of the native protein. By thermal destabilization to the molten globule state, access is given to two domains with strong hydrophobic character. On further heating the hydrophobic domains degenerate; however, the temperature of destabilization is different for the two domains. A comparable evolution of the hydrophobic behavior is observed for apo- and Ca(2+)-BLA, but the destabilization steps of Ca(2+)-BLA occur at higher temperatures than those of apo-BLA. Also, it has not been reported earlier that, at a moderate Ca2+ concentration (2 mM), Ca2+ remains associated with BLA after the thermally induced destabilization of its native tertiary structure. At 70 degrees C, a partially unfolded state of Ca(2+)-loaded BLA is obtained.