Studies on the binding of copper to dopamine beta-monooxygenase and other proteins using the Cu2+ ion-selective electrode.

The binding of Cu2+ to native and copper-free dopamine beta-monooxygenase has been investigated by potentiometric titrations using a Cu2+-selective electrode. Stoichiometric formation constants have been determined from regression analysis of the resulting titration curves. The results establish a stoichiometry of four high-affinity binding sites for Cu/+ (log Kf approximately 11) per enzyme tetramer, and more binding sites of lower affinity (log Kf approximately 5-7). The data for binding of the first four Cu2+ to the enzyme tetramer indicate interactions in the binding to the sites. Bovine serum albumin, metal-free carbonic anhydrase, and ovotransferrin have also been titrated with Cu2+, and the formation constants of both high-affinity binding sites and other sites have been determined. The stoichiometry of one high-affinity binding site of Cu2+ for carbonic anhydrase (log Kf approximately 10-12) and two sites for ovotransferrin (log Kf approximately 11) agree with the reported metal binding properties of these proteins. The number of high-affinity binding sites for bovine serum albumin was pH dependent.