Reaction of human .alpha.2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure

Abstract

Human .alpha.2-macroglobulin (.alpha.2M) half-molecules were prepared by limited reduction and alkylation of the native protein. Reaction with plasmin resulted in nearly quantitative cleavage of the half-molecule MW .apprx. 180,000 subunits into MW .apprx. 90,000 fragments. Subunit cleavage was significantly less complete when plasmin was reacted with .alpha.2M whole molecules. The plasmin and trypsin binding capacities of the 2 forms of .alpha.2M were compared by using radioiodinated proteases. .alpha.2M half-molecules bound an equivalent number of moles of plasmin or trypsin. Native unreduced .alpha.2M bound only half as much plasmin as trypsin. These data are consistent with the hypothesis that the 2 protease binding sites are adjacent in native .alpha.2M. .alpha.2M half-molecule-plasmin complexes reassociated less readily than half-molecule-trypsin complexes, supporting this interpretation. The frequency of covalent bond formation between plasmin and .alpha.2M was considerably higher than that previously observed with other proteases. Of the plasmin that reacted with .alpha.2M whole molecules or half-molecules, .apprx. 80-90% became covalently bound. The reactivities of purified .alpha.2M-plasmin complexes were compared with small and large substrates. Equivalent kcat/Km values were determined at 22.degree. C for the hydrolysis of H-D-Val-Leu-Lys-p-nitroanilide dihydrochloride by whole molecule-plasmin complex and half-molecule-plasmin complex (40 mM-1 s-1 and 39 mM-1 s-1, respectively, compared with 66 mM-1 s-1 determined for free plasmin). Complexes of plasmin and the 2 different forms of .alpha.2M digested fibrinogen at comparable rates (slowly compared with free plasmin); however, the half-molecule-plasmin complex demonstrated increased reactivity with soybean trypsin inhibitor. .alpha.2M half-molecule-plasmin complex cleared rapidly from the circulation of mice, reflecting receptor binding and endocytosis.