Equilibrium binding of antithrombin and thrombin to EP compounds. (A) Relative protein fluorescence changes (F_obs/F_o) accompanying the stepwise addition of EP42675 (open symbols) or EP217609 (closed symbols) to 500nM antithrombin in I 0.05 sodium phosphate buffer (○, ●) or to 200nM antithrombin in I 0.15 HEPES plus Ca²⁺ buffer (▵, ▴) both at pH 7.4, 25°C, are plotted as a function of the molar ratio of EP compound to antithrombin. Solid lines indicate fits to the quadratic equilibrium binding equation from which the binding stoichiometry, the K_D (fixed at 0.5nM for I 0.05 titrations) and the maximal fluorescence change were derived. (B) Relative changes in p-aminobenzamidine fluorescence (F_obs/F_o) accompanying the stepwise addition of EP42675 (○) or EP217609 (●) to 200nM thrombin plus 100μM p-aminobenzamidine are plotted as a function of the molar ratio of EP compound to thrombin. Titrations were fit by the quadratic equilibrium binding equation in which K_D was fixed at a value of 0.1nM based on measurements of K_I and the competitive effect of probe binding to obtain values for the binding stoichiometry and maximal fluorescence change.