FIGURE 4.
Effect of active site labeling of APC with FPR-CH2Cl on competitive binding of APC and OG-FXa to native FVa. The fractional fluorescence change (ΔF/Fo) of a mixture of 1.2 nm OG-FXa and 5.8 nm FVa as a function of the total concentration of APC(S360A) (●) or FPR-APC (○) in the presence of 50 μm phospholipid vesicles (20:60:20, DOPS:DOPC:DOPE) is shown. The solid lines represent fits of the data by the cubic competitive binding equation with fixed parameters of n = 0.82 ± 0.18 FVa/OG-FXa (mol/mol), KP = 0.14 nm, and fitted parameters, m = 1.4 ± 0.4 APC(S360A)/FVa (mol/mol), KC = 0.17 ± 0.09 nm, and ΔFmax/Fo = 38 ± 2%. For FPR-APC, m was fixed at 1, KC = 36 ± 9 nm, and ΔFmax/Fo = 36 ± 1%. The error bars represent the 95% confidence interval. Fluorescence titrations were performed and analyzed as described under “Experimental Procedures.”