ELECTROCHEMICAL STUDIES OF THE INTERFACIAL BEHAVIOR OF INSULIN

The interfacial behavior of insulin and chain A and chain B of insulin was investigated at the platinum electrode in a phosphate buffer, pH 7.0, using cyclic voltammetry. The enthalpy of adsorption, Delta H-ADS , calculated from a linear van't Hoff relationship over the temperatur e range 299 to 333 K gave values of -22 +/- 1, -17 +/- 1, and -9 +/- 1 kJ mol(-1) for insulin, chain B and chain A, respectively, Above thes e temperatures denaturation of insulin occurs, and for all three molec ules the surface adsorption measured by the surface charge densities s howed an immediate decrease followed by a slight increase. The surface concentrations of insulin of 2.9 +/- 0.2 mg m(-2) at 299 K and 3.2 +/ - 0.3 mg m(-2) at the physiological temperature of 310 K agreed well w ith the calculated value determined from geometrical dimensions, Simil ar calculations from experimental surface charge densities for chain A and chain B indicated that a more efficient packing prevailed with th e individual polypeptides. From a consideration of the mechanism of ad sorption based on cyclic voltammetric measurements, an estimation of t he number of carboxylate groups on insulin was determined to be 6 +/- 2, which agrees with the known number of acidic residues on the protei n. Similar calculations for chain A gave 2 +/- 0.4, which indicates th at this peptide remains as a monomer in the phosphate buffer, However, the value obtained for chain B under similar experimental conditions was 6 +/- 2, indicating that this peptide appears to dimerize in the p hosphate buffer. Dimer formation of insulin is known to occur through hydrophobic interactions and four hydrogen bonds between the B chains. (C) 1996 Academic Press, Inc.