Comparative studies on native and elastase-inactivated α1-protease inhibitor ( α1-antitrypsin)

Abstract

Purified α 1-protease inhibitor was reacted with a 1.3-fold molar excess of swine pancreatic elastase and incubated at 37°C for 30 min. Total inactivation of the inhibitor occurs under these conditions. Inactivated α 1-protease inhibitor was isolated from the reaction mixture by chromatography on DEAE-cellulose. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the preparation to be homogeneous. The amino acid composition of native ( M r 54 000) and inactivated ( M r 49 000) α 1-protease inhibitor were practically identical; sialic acid residues were unchanged at 6 per mol. Carbohydrate analyses showed a net loss of 8 N-acetylglucosamine and 10 hexose residues per mol of inactivated inhibitor when compared to the native protein. The weight loss which occurs in α 1-protease inhibitor die to inactivation by elastase (54 000 vs. 49 000) is probably due primarily to the loss of carbohydrate residues. Electrophoresis of cyanogen bromide fragments in sodium dodecyl sulfate-ureapolyacrylamide gel produced nine distinct zones for the native inhibitor, eight of which were common to the inactivated protein. The missing zone in the elastase-inactivated inhibitor CNBr preparation was strongly periodic acid-Schiff positive, suggesting that the carbohydrate moiety of this glycopeptide is cleaved when the native protein is inactivated by elastase. Electrophoretic mobility determinations, at pH 8.6, gave −5.5 · 10 −5 cm 2 · V −1 · s −1 for the native protein and −5.6 · 10 −5 cm 2 · V −1 · s −1 for the inactivated inhibitor. The inactivated protein retained its ability to produce multiple zones upon isoelectric focusing with isoelectric points varying between 4.53 and 4.75 (av. 4.61). The average isoelectric point for the native inhibitor was 4.45 (4.38−4.51).