Figure 5. Rapid kinetics of ligand binding to the trypsin-like proteases thrombin, FXa, and aPC.

Rates of approach to equilibrium, k_obs, determined from stopped-flow fluorescence measurements of binding of PABA (red), KCl (magenta), NaCl (blue), VPR (orange), and CaCl₂ to (A) thrombin, (B) FXa, and (C) aPC. Reaction conditions were as follows: (A) 5 mM Tris, 0.1% PEG8000, pH 8.0 at 15 °C, with ionic strength kept constant at 400 mM with ChCl; (B) 50 mM Tris, 0.1% PEG8000, pH 8.0 at 15 °C, with no ChCl used due to specific inhibition of FXa by choline; (C) 5 mM Tris, 0.1% PEG8000, pH 8.0 at 15 °C, with ionic strength kept constant at 800 mM with ChCl. Solid lines were drawn according to the equation k_r+k_−r/{1+[L]/K_d} for PABA and KCl in thrombin, PABA and NaCl in FXa, and NaCl in aPC, or according to eq. 2 in the text. Best-fit parameters for thrombin binding to ligand (A) are: (PABA) k_r=70±10 s⁻¹, k_−r=340±10 s⁻¹, K_d=52±8 μM; (K⁺) k_r=60±10 s⁻¹, k_−r=340±10 s⁻¹, K_d=19±2 mM; (Na⁺) k_r=60±10 s⁻¹, k_−r=340±10 s⁻¹, k_on=3.2±0.3 10⁴ M⁻¹s⁻¹, k_off=130±20 s⁻¹; (VPR) k_r=80±10 s⁻¹, k_−r=340±10 s⁻¹, k_on=1.7±0.1 10⁷ M⁻¹s⁻¹, k_off=2.4±0.2 s⁻¹. Best-fit parameters for FXa binding to ligand (B) are: (PABA) k_r=48±36 s⁻¹, k_−r=327±29 s⁻¹, K_d=44±2 μM; (Na⁺) k_r=43±4 s⁻¹, k_−r=240±20 s⁻¹, K_d=10±1 mM. Best-fit parameters for aPC binding to ligand (C) are: (Na⁺) k_r=25±1 s⁻¹, k_−r=24±4 s⁻¹, K_d=9±1 mM; (Ca²⁺) k_r=20±1 s⁻¹, k_−r=25±4 s⁻¹, k_on=16±1 10³ M⁻¹s⁻¹, k_off=5.0±0.3 s⁻¹. Because k_−r was not well determined in FXa binding to Na⁺, it was restricted by the values derived from PABA binding. A similar procedure was used for k_on for Ca²⁺ binding to aPC. To facilitate comparison, the values of [L] for each ligand are expressed as the fraction of maximal ligand used.