INTRINSIC STOICHIOMETRIC EQUILIBRIUM-CONSTANTS FOR THE BINDING OF ZINC(II) AND COPPER(II) TO THE HIGH-AFFINITY SITE OF SERUM-ALBUMIN

Intrinsic stoichiometric equilibrium constants were determined for zin c(II) and copper(II) binding to bovine and human serum albumin. Data w ere obtained from equilibrium dialysis experiments. Metals were presen ted to apoprotein as metal chelates in order to avoid metal hydrolysis and to minimize nonspecific metal-protein interactions. Scatchard ana lysis of the binding data indicated that the high affinity class for b oth zinc and copper was comprised of one site. Results of binding expe riments done at several pH values suggested that while both histidyl a nd carboxyl groups appear to be involved in copper binding, histidyl r esidues alone were sufficient for zinc binding. These amino acid resid ues were used in combination to model several binding sites used in th e formulation of equilibria expressions from which stoichiometric cons tants were calculated. The log10K for bovine serum albumin were calcul ated to be 7.28 for Zn(II) and 11.12 for Cu(II). Those for human serum albumin were determined to be 7.53 and 11.18 for Zn(II) and Cu(II), r espectively. These constants were used in equilibria to simulate speci ation of metal-albumin and metal-chelator and to illustrate relative b inding affinities. This comparison of binding strengths was possible o nly through the calculation of an intrinsic stoichiometric binding con stant.