The serpin molecule has evolved an unusual mechanism of inhibition, involving an exposed reactive center loop (RCL) and conformational change to covalently trap a target protease. Successful inhibition of the protease is dependent on the rate of serpin-protease association and the efficiency with which the RCL inserts into β-sheet A, translocating the covalently bound protease and thereby completing the inhibition process. This chapter describes the kinetic methods used for determining the rate of protease inhibition (k(a)) and the stoichiometry of inhibition. These kinetic variables provide a means to examine different serpin-protease pairings, assess the effects of mutations within a serpin on protease inhibition, and determine the physiologically cognate protease of a serpin.
Copyright © 2011 Elsevier Inc. All rights reserved.