Studies on the elastase-serum protein interaction. III. The elastase inhibitors of swine serum with emphasis on the elastase- α2-macroglobulin interaction

Abstract

Impregnation of agar gel electrophoretograms of swine serum with elastase (pancreatopeptidase E, EC 3.4.4.7) followed by overlaying with elastin particles in agar revealed that swine serum contains three protein inhibitors of elastase, an α 0/ ρ 1-globulin, and α 2-globulin and a β 1-globulin. The α 0/ ρ 1- and α 2-globulins are analogous to the α 1-antitrypsin and α 2-macroglobulin of human serum while the β 1- globulin, the principal elastase inhibitor of swine serum, has no apparent human serum counter part. The molecular weights and electrophoretic mobilities of the α 0/ ρ 1-globulin, α 2-macroglobulin and the β 1-globulin were found, respectively, to be: 72 000, −6.1·10 −5 cm 2·V −1·s −1; 960 000 (ref. 15), −4.1·10 −5 cm −5·V −1·s −1; 83 600, −3.2·10 −5 cm 2·V −1·s −1. 1 ml of swine serum was capable of binding 0.6 mg of swine elastase. Swine α 2-macroglobulin was purified from swine whole serum by fractional precipitation with (NH 4) 2SO 4 followed by ultracentrifugation, Sephadex G-200 gel filtration and DEAE-cellulose column chromatography. An inhibition curve (elastase activity vs increasing amounts of α 2-macroglobulin) revealed that 1.13 mole of α 2-macroglobulin (mol. wt 960 000) was capable of binding 1.0 mole of elastase (mol. wt 25 000). The dissociation constant for the α 2-macroglobulin elastase complex was 4.5·10 −9. pH-stat experiments with α 2-macroglobulin-elastase mixtures (molar ratio α 2-macroglobulin:elastase > 1.0) revealed an appreciable cleavage of peptide bonds within 16 h. Peptide bond cleavage was also demonstrated by gel filtration of 48-h digests on Sephadex G-50 and G-200 and by peptide mapping.