Inhibition of Neutrophil Elastase by Alpha-1-Proteinase Inhibitor Oxidized by Activated Neutrophils

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Abstract
The present study was aimed at testing whether neutrophil-oxidized .alpha.1-proteinase inhibitor (.alpha.1 PI) is a slow-binding inhibitor of neutrophil elastase like N-cholorosuccinimide-oxidized .alpha.PI or whether it does not inhibit this enzyme at all as currently thought, .alpha.1PI was reacted with phorbol-myristate-acetate-activated neutrophils and isolated from the oxidation medium by fast protein liquid chromatography on an anion exchange column. When sufficient time was allowed for oxidized .alpha.1PI to react with neutrophil elastase (2 h), enzyme-inhibitor complex formation could be demonstrated by three means: (1) inhibition of elastase activity, (2) detection of the complex using an enzyme-linked immunosorbent assay specific for the native .alpha.1PI-elastase complex, and (3) SDS-polyacrylamide gel electrophoresis, which evidenced a complex with a molecular weight of 80,000. Porcine pancreatic elastase was not inhibited. Neutrophil-oxidized .alpha.1PI behaved as an irreversible inhibitor of neutrophil elastase, as revealed by the kinetic analysis of the inhibition reaction. The rate constant for the inhibition of neutrophil elastase by neutrophil-oxidized .alpha.1PI (0.76 .+-. 0.22 .times. 104 M-1 s-1) was close to that for the inhibition of the enzyme by N-chlorosuccinimide-oxidized .alpha.1PI (0.96 .+-. 0.24 .times. 104 M-1 s-1), but it was more than three orders of magnitude lower than that for the reaction of native .alpha.1PI with neutrophil elastase. Both native and oxidized .alpha.1PI were temporary elastase inhibitors: the enzyme slowly and spontaneously escaped the complex with formation of an inactive .alpha.1PI derivative with a molecular weight of 49,000. This reaction was followed for 1 wk for SDS-polyacrylamide gel electrophoresis and elastase activity measurements in the presence or absence of .alpha.2-macroglobulin. Neutrophil elastase was released from its complex with neutrophil-oxidized .alpha.1PI at a rate (kdeg = 1.1 .times. 10-5 s-1, half-time = 0.7 day) somewhat faster than that of its release from its complex with native .alpha.1PI(kdeg = 2 .times. 10-6 s-1, half-time = 4 days). In conclusion, the major difference between native and neutrophil-oxidized .alpha.1PI is the slow rate at which the latter inhibits neutrophil elastase. The expression "oxidative inactivation of .alpha.1PI" is therefore misleading. Oxidized .alpha.1PI reacts probably too slowly with neutrophil elastase to play an emphysema-preventing function. It might control physiologic proteolytic processes catalyzed by neutrophil elastase (e.g., during neutrophil migration) by allowing elastase to cleave substrate for a limited period of time before irreversible inhibition takes place.