Conformational aspects of the Cu2+ binding to alpha-lactalbumin. Characterization and stability of the Cu-bound state.

By circular dichroism experiments the existence of a typical Cu2(+)-bound state is demonstrated for bovine- and for goat alpha-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 net effect of Cu2+ ions on the protein conformation. The Cu2(+)-bound state, characterized by a vanishing tertiary structure and a substantial loss of secondary structure, clearly differs from the well-known Ca2(+)-, apo-, and acid conformers. At room temperature, the Cu2+ binding has already decreased the alpha-helix content of bovine alpha-lactalbumin to the extent that further unfolding by thermal or guanidine hydrochloride denaturation behaves in a non-cooperative way. Since for goat alpha-lactalbumin the Cu2+ binding to His-68 is much less important than for bovine alpha-lactalbumin, we observe a somewhat different conformational behaviour for goat alpha-lactalbumin. The results of this conformational circular dichroism study are confirmed by isothermal calorimetric data.