Figure 4
Relationship between the apparent first-order rate and the concentration of tight binding inhibitor sculptin. (A) Typical progress curves for hydrolysis of 15 µM S-2238 chromogenic substrate by 0.1 nM thrombin in absence (trace a) and presence of sculptin (trace b, 10 pM; trace c, 30 pM; trace d, 70 pM; trace e, 100 pM; trace f, 200 pM and trace g, 500 pM) in 50 mM phosphate buffer containing 150 mM NaCl and 0.1% PEG 6000, pH 7.4 at 37 °C. Reactions were started with addition of thrombin to the mixture containing sculptin and S-2238. (B) Steady-state velocity of thrombin with respect to sculptin concentration. The inset shows the determination of the apparent dissociation constant, Ki*, from steady state velocities. The data was fitted into a linear regression to obtain Ki. (C) Calculation of the dissociation constant from kobs. Progress curves were produced with 15 µM S-2238, 0–50 pM sculptin, and 100 pM thrombin. The apparent first-order rate constant was calculated using a nonlinear regression fit, where the intercept and slope is kon and koff respectively. The experimental condition of (B) and (C) is the same as (A). The data in (B) and (C) correspond to the mean ± standard deviation values acquired in five independent experiments.
