The first detailed kinetic study of in vitro reactions between serum albumin and the secondgeneration gold drug 3,4,glucopytanosato-S-) gold(I)] and its tri-i-propylphosphine analogue, .iPr3PAuSATg, are reported. The reactions were investigated using Penefsky spun columns and NMR saturation transfer kinetics.Based on the Penefsky column data, the binding of the Et3PAuSATg to AIbSH (0.600 mM) was complete when gold concentrations were limiting: 0.093, 0.151, and 0.225 mM. The reaction is biphasic. The fast phase is defined by a first order rate constant, (k 2.94 _+ 0.24 x 10 -2 sec"1) and accounts for 95% of the Au(I) bound. This phase is first order with respect to albumin, and zero order with respect to auranofin. A minor, slower step (k 2 2.26 _+ 0.26 x 10 -3 sec"1), which accounts for only 5% of the reaction, is also first order with respect to albumin, and zero order with respect to auranofin. For .iPr3PAuSATg, only the first step was observed, k 1.4 +_ 0.1 x 10 -2 s "1 and is first order in albumin and independent of iPr3PAuSATg concentration. 31p.NMR saturation transfer experiments utilizing the auranofin analogue, .iPr3PAuSATg, under equilibrium conditions with excess .iPr3PAuSATg and ATgSH yielded second order rate constants for both the forward (1.0 x 102 M " sec"1) and the reverse (3.9 x 101 M "1 sec"1) directions. A multi-step mechanism involving a conformationally altered albumin species to which gold binds was developed using the steady-state approximation. The mechanism accounts for the different reaction orders observed under the two set of conditions. A rate determining conformational change on the albumin governs the reaction as monitored by the Penefsky columns. A second order reaction of R3PAuSATg at Cys-34 is observed under the NMR conditions. These results are the first quantitative determination of auranofin-albumin reaction kinetics and the first mechanistic study of the reaction.A novel binding mechanism, association of auranofin in the pocket of albumin-disulfide species can be detected by Penefsky and HummeI-Dreyer gel chromatographic techniques, but not by conventional gel-exclusion chromatography. This complex albumin-auranofin complex is weaklybound and readily dissociates.

The first detailed kinetic study of in vitro reactions between serum albumin and the secondgeneration gold drug Auranofin [Et3PAuSATg triethylphosphine-(2,3,4,6-tetra-O-acetyl-l-8-Dglucopytanosato-S-) gold(I)] and its tri-i-propylphosphine analogue, .iPr3PAuSATg, are reported. The reactions were investigated using Penefsky spun columns and NMR saturation transfer kinetics. Based on the Penefsky column data, the binding of the Et3PAuSATg to AIbSH (0.600 mM) was complete when gold concentrations were limiting: 0.093, 0.151, and 0.225 mM. The reaction is biphasic. The fast phase is defined by a first order rate constant, (k 2.94 _+ 0.24 x 10-2 sec"1) and accounts for 95% of the Au(I) bound. This phase is first order with respect to albumin, and zero order with respect to auranofin. A minor, slower step (k 2 2.26 _+ 0.26 x 10-3 sec"1), which accounts for only 5% of the reaction, is also first order with respect to albumin, and zero order with

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