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 A from a cationic benzamidine function that competitively inhibits C1s esterase and protease activity. Purified human C1s 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 C1s, 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 C1s at several mCP(PBA)-F concentrations followed a rectangular hyperbolic function preducted for affinity labeling kinetics (K L = 87.3 µM, k 2 = 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.