Effect of various divalent cations on the reversal of the AT-heparin inhibition of FXa by HRPII. (A) Effect of various cations and Zn²⁺ counterions. Heparin (0.01 U/mL), AT (100nM), with or without HRPII (300nM) were added to a fixed amount of FXa (5nM) in buffer without divalent ions, or buffer containing the indicated salt (2mM for MgCl₂ and 15μM for the rest). The resulting mixture was incubated for 1 minute at room temperature, and the FXa activity remaining was then determined by amidolytic assay using S-2222 as described in “Amidolytic assays.” Note that AT-heparin inhibition of FXa is enhanced by Mg²⁺. Assays were done in triplicate, and results are the mean ± SE. (B) Zn²⁺ titration for determination of K_d for Zn²⁺ binding to HRPII. The assay was performed as described in panel A in the presence of HRPII, using various concentrations of zinc acetate (0-160μM), at fixed heparin (0.01 U/mL), AT (100nM), HRPII (300nM), FXa (5nM), and CaCl₂ (5mM).