FIGURE 3.

Pre-steady state kinetics of hirudin binding to prethrombin-2 showing the relaxation derived from the single exponential fit to the kinetic traces for hirudin binding to prethrombin-2 in the presence of excess hirudin (A) or prethrombin-2 (B). The continuous line was drawn according to Equation 2 in the text (A) or a modified version of Equation 1 with k_on replaced by the expression k_onk₁₂/(k₁₂ + k₂₁) to account for the fraction of prethrombin-2 in the E form (B). Both data sets were analyzed simultaneously with proper weighting to yield best-fit parameters: k₁₂ = 21 ± 3 s⁻¹, k₂₁ = 200 ± 100 s⁻¹, k_off = 3.1 ± 0.1 s⁻¹, k_on = 4 ± 2 μm⁻¹ s⁻¹. Experimental conditions are: 50 mm Tris, 0.1% PEG8000, 400 mm ChCl, pH 8, at 15 °C.