Conformational aspects of the Cu 2+ binding to α-lactalbumin. Characterization and stability of the Cu-bound state

Abstract

By circular dichroism experiments the existence of a typical Cu 2+-bound state is demonstrated for bovine- and for goat α-lactalbumin. As in the near-UV region an important ligand to metal charge-transfer band overlaps with the aromatic band of the protein, a subtraction method is developed in order to determine the nett effect of Cu 2+ ions on the protein conformation. The CU 2+-bound state, characterized by a vanishing tertiary structure and a substantial loss of secondary structure, clearly differs from the well-known Ca 2+-, apo-, and acid conformers. At room temperature, the Cu 2+ binding has already decreased the α-helix content of bovine α-lactalbumin to the extent that further unfolding by thermal or guanidine hydrochloride denaturation behaves in a non-cooperative way. Since for goat α-lactalbumin the CU 2+ binding to His-68 is much less important than for bovine α-lactalbumin, we observe a somewhat different conformational behaviour for goat α-lactalbumin. The results of this conformational circular dichroism study are confirmed by isothermal calorimetric data.