Fig. 3.
Kinetic analysis of the time-dependence of BDGA binding to Hsp90α. BDGA binding to Hsp90α was measured by monitoring the fluorescence anisotropy of BDGA (10 nM) as a function of incubation time in the presence of varying concentrations of the protein. BDGA unbound in solution and bound to Hsp90α has approximate anisotropy values of 0.04 and 0.17, respectively. Data were fit to a pseudo-first-order rate equation, Eq. 8, to determine kobs values. (A) [Hsp90]: 1.5 μM (▾), 1.25 μM (▿), 0.90 μM (▴), 0.70 μM (▵), 0.50 μM (■), 0.30 μM (□), 0.10 μM (●), 0 μM (○). (B) [Hsp90]: 0.10 μM (▴), 0.075 μM (▵), 0.050 μM (■), 0.025 μM (□), 0.0125 μM (●), 0 μM (○). (C) Replot of kobs vs. [Hsp90]. Data fit to the equation for a two-step protein–ligand-binding model, Eq. 1. Kinetic constants derived from the data are summarized in Table 2.