FIGURE 5. Kinetic and end point titrations of [5F]Hir-(54 – 65)( ${\text{SO}}_3^{-}$) binding to MzT(−F1) in the absence and presence of Na⁺.

A, kinetic titration of fluorescence changes (ΔF/F_o) of 20 nM [5F]Hir-(54 – 65)( ${\text{SO}}_3^{-}$) during activation of Pre 1 at concentrations ranging from 58 nM to 1000 nM, initiated by 2 units/ml ecarin in buffer containing NaCl (○). The lines represent the fit of the fluorescence data with the parameters listed in Table 1. B, analysis of the reaction end points as a function of the total Pre 1 concentration ([Pre 1]_o). The line represents the fit by the quadratic binding equation with the parameters listed in Table 1. C, kinetic titrations for activation of 116 –1450 nM Pre 1 in buffer containing ChCl (○) otherwise as described in A, along with the simultaneous fit with the parameters listed in Table 1. D, end point titration for the reactions in buffer containing ChCl as described in B. Reactions were performed and analyzed as described under “Experimental Procedures.”