KINETICS AND MECHANISM OF THE REACTION BETWEEN SERUM-ALBUMIN AND AURANOFIN (AND ITS ISOPROPYL ANALOG) IN-VITRO

The first detailed kinetic analysis and mechanistic interpretation of the reactions between serum albumin and the second-generation gold dru g Auranofin [Et(3)PAuSATg = ,3,4,6-tetra-O-acetyl-1-beta-D-glucopyrano sato-S-) gold(I)] and its triisopropylphosphine analogue, iPr(3)PAuSAT g, in vitro are reported. The reactions were investigated using Penefs ky spun columns and NMR saturation transfer methods. Under the Penefsk y chromatography conditions with 0.4-0.6 mM albumin and a wide range o f Et(3)PAuSATg concentrations, the reaction is biphasic. The fast phas e is apparently first order in albumin with a rate constant [k(1) = 3. 4 +/- 0.3 x 10(-2) s(-1)] that decreases slightly in magnitude and bec omes intermediate in order at low gold concentrations, [Et(3)PAuSATg] [AlbSHH]; it accounts for similar to 95% of the Au(I) that binds. A mi nor, slower step [k(2) = 2.3 +/- 0.3 x 10(-3) s(-1)], which accounts f or only 5% of the reaction, is also first order with respect to albumi n, and zero order with respect to auranofin. For iPr(3)PAuSATg, only t he first step was observed, k(1) = (1.4 +/- 0.1) x 10(-2) s(-1), and i s first order in albumin and independent of the iPr(3)PAuSATg concentr ation. P-31-NMR saturation transfer experiments utilizing iPr(3)PAuSAT g, under equilibrium conditions, yielded second-order rate constants f or both the forward (1.2 x 10(2) M(-1) s(-1)) and the reverse (3.9 x 1 0(1) M(-1) s(-1)) directions. A multistep mechanism involving a confor mationally altered albumin species was developed. Albumin domain IA op ens with concomitant Cys-34 rearrangement, allowing facile gold bindin g and exchange, and then closes. In conjunction with the steady-state approximation, this mechanism accounts for the different reaction orde rs observed under the two set of conditions. The rate-determining conf ormational change of albumin governs the reaction as monitored by the Penefsky columns. Rapid second order exchange of R(3)PAuSATg at the ex posed Cys-34 residue is observed under the NMR conditions. The mechani sm predicts that under physiological conditions where [Et(3)PAuSATg] i s 10-25 mu M, the reaction will be second order and rapid with a rate constant of 8 +/- 2 x 10(2) M(-1) s(-1). The Penefsky spun columns rev ealed a previously unreported and novel binding mechanism, association of auranofin in the pocket of albumin-disulfide species, which was co nfirmed by Hummel-Dreyer gel chromatographic techniques under equilibr ium conditions. This albumin-auranofin complex (AlbSSR-Et(3)PAuSATg) i s weakly hound and readily dissociates during conventional gel exclusi on chromatography.