Figure 6. Determination of IC₅₀ values based on dose response plots of fractional velocity as a function of different tetra mutant CrSPI-1-D1 concentration.

OD₄₀₅ was monitored based on hydrolysis of 0.1 mM S2238 (H-D-Phenylalanyl-L-pipecolyl-Larginine-p-nitroaniline dihydrochloride) and formation of colored product, p-nitroaniline, by 4.5 nM thrombin in a total reaction volume of 200 µL. V₀ and V_i are the initial velocities in the absence and presence of tetra mutant CrSPI-1-D1, respectively and were calculated from Beer-Lambert's Law. The tetra mutant CrSPI-1-D1 concentration predicted to block 50% of the activity of a fixed concentration of thrombin was obtained on a graphical plot of V_i/V₀ versus inhibitor concentration.