Isolation and Characterization of Porcine Leukocyte Elastase Leukocyte Elastase-Inhibitor Complexes in Porcine Blood, II1)

Summary: Porcine leukocyte elastase1) was purified from granulocytes by chelating chromatography on copper chelate Sepharose and by ion exchange chromatography on CM-Sepharose. Thus an enzyme prepara-tion with a specific activity (substrate: MeOSuc(Ala)2ProValNan)2) of 89.3 U/mg protein was obtained. Dodecyl sulphate gel electrophoresis revealed one protein band corresponding to a molecular mass of 27 kDa. The amino acid composition was determined and isoleucine was identified äs the only N-terminal amino acid residue. The bimolecular velocity constant for the Inhibition by diisopropyl fluorophosphate was determined äs 2000 ( I x mol"1 min"1. The dissociation constants, Ki? of the complexes of porcine leukocyte elastase with r various Inhibitors were calculated. The kinetic constants for the elastase-catalysed hydrolysis of MeOSuc-j. (Ala)2ProValNan, Suc(Ala)2ValNan and Suc(Ala)3Nan were determined, äs well äs the kinetic constants of the inactivation of leukocyte elastase by active site mapping reagents. Detergents such äs Triton X-100, Tween 20 and Brij 35, äs well äs porcine serum albumin, activated the procine leukocyte elastase preparation. Isolierung und Charakterisierung der Elastase aus Leukocyten des Schweins Leukocyten-ai-Proteinaseinhibitor-Komplexe in Schweineblut, II. Mitteilung Zusammenfassung: Elastase2) aus Schweineleukocyten wurde mit Hilfe der Chelatchromatographie an Kup