Affinity Labeling of C1s̄ with an Extended Reagent

The reagent m-[o-(2-chloro-5-sulfonylfluorphenylureido) phenoxybutoxybenzamidine [mCP(PBA)-F] is an extended affinity labeling reagent in which the reactive sulfonylfluoride is 17.1 Å from a cationic benzamidine function that competitively inhibits C1s̄ esterase and protease activity. Purified human C1s̄ with high specific esterolytic activity was selectively and irreversibly inactivated with mCP(PBA)-F. At 16.6 µM mCP(PBA)-F and 22.1 µM C1s̄, the time course of inactivation followed pseudo first order kinetics (k_L = 2.45 × 10^-3 sec^-1, 23°C). The initial rate of inactivation of C1s̄ at several mCP(PBA)-F concentrations followed a rectangular hyperbolic function preducted for affinity labeling kinetics (K_L = 87.3 µM, k₂ = 3.02 × 10^-3 sec^-1). The hydrolyzed affinity labeling reagent lacking the reactive sulfonylfluoride group [mCP(PBA)-OH] was a strong competitive inhibitor (K_I = 6.82 µM vs 0.63 mM for benzamidine alone) implicating active site binding.