Oxidized mucus proteinase inhibitor: A fairly potent neutrophil elastase inhibitor

Abstract

N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1·s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant k(ass.) = 2.6 x 105 M-1·s-1, dissociation rate constant k(diss.) = 2.9 x 10-3 s-1 and equilibrium dissociation constant K(i) = 1.1 x 10-8 M. Comparison with the native inhibitor indicates that oxidation decreases k(ass.) by a factor of 18.8 and increases k(diss.) by a factor of 6.4, and therefore leads to a 120-fold increase in K(i). Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than K(i), i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 μM elastase is fully inhibited by 5-6 μM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not.