FIGURE 5.

Hirugen binding to thrombin wild type (left) and WE (right) measured by isothermal titration calorimetry. The top panel shows the heat exchanged in each individual titration for the thrombin sample (lower trace) and the hirugen buffer control (upper trace, left). The bottom panel is the integration of the data to yield the overall heat exchanged as a function of the ligand/protein molar ratio. Experimental conditions are: 5 mm Tris, 0.1% polyethylene glycol 8000, 200 mm NaCl, pH 8.0, 25 °C. The enzyme and hirugen concentrations are: 10 and 105 μm (thrombin wild type); 12 and 220 μm (WE). Titration curves were fit using the Origin software of the iTC200, with best-fit parameter values: thrombin wild type, K = 7.4 ± 0.1 10⁶ m⁻¹, ΔG = −9.4 ± 0.1 kcal/mol, ΔH = −14.5 ± 0.1 kcal/mol, TΔS = −5.1 ± 0.1 kcal/mol; WE, K = 2.1 ± 0.5 10⁶ m⁻¹, ΔG = −8.6 ± 0.1 kcal/mol, ΔH = −14.0 ± 0.1 kcal/mol, TΔS = −5.4 ± 0.1 kcal/mol. The value of the stoichiometric constant, N, was 0.97 ± 0.01 for thrombin wild type and 1.02 ± 0.01 for the WE mutant.