Figure 1. Binding interactions of ZMC1, DBD, and zinc.

A, titration of ZMC1 and ZnCl₂. Extrapolation of the linear phases shows 0.51 ± 0.01 equivalents of Zn²⁺ at saturation. Inset shows normalized absorbance spectra for ZMC1 alone (black) and ZMC1-Zinc complex (red). B, measurement of ZMC1-zinc complex dissociation rate. Solutions of ZnCl₂ and ZMC1 were mixed 1:1 with 2 mM EDTA to the final concentrations indicated (written ZnCl₂/ZMC1). The traces are an average of 3-4 injections and when fit to a single exponential, yield nearly identical rates, which we combine to report a k_off of 2.6 ± 0.1 s⁻¹. C, structures of ZMC1 and A6 and their K_d's for Zn²⁺. D, determination of the K_d of R175H DBD for Zn²⁺.R175H DBD (5 μM, 0.6 equivalents Zn²⁺ co-purified) was incubated with increasing concentrations of PAR to compete for the available Zn²⁺. The concentration of PAR₂Zn²⁺ complex was determined by absorbance, and the parallel mass action and mass conservation equations solved to determine K_d. K_d's from the 2-site model are 2.1 ± 0.8 nM for the tighter site (K_d1) and below the detectable limit for the weaker site (K_d2), respectively.