Covalent Thrombin-α²-Macroglobulin Complexes. Evidence for Bivalent Cross-Linking of Inhibitor Chains by a Single Enzyme Molecule

Complexes formed between thrombin and α₂-macroglobulin (α₂M) were studied by polyacrylamide gel electrophoresis. The results provide evidence for the existence of a recently proposed novel enzyme-inhibitor species in which a single thrombin molecule forms two or more covalent bonds to two or more different α₂M chains. At least one of several slowly migrating bands (greater than 375K on nonreduced gels) that have previously been observed in the literature but not well characterized can be assigned to the new species. The involvement of the lysyl amino groups of thrombin is shown by the observation that methylation of these groups reduces the higher molecular weight bands. In addition, increasing the thrombin:α₂M ratio causes a relative decrease in the higher molecular weight species, suggesting that these complexes arise by intramolecular reactions that are susceptible to competition by solution thrombin. The data provide support for our previous proposal [Wang, D., Yuan, A., & Feinman, R. D. (1983) Ann. N.Y. Acad. Sci. 421, 90-97] that the 260K band seen in reduced gels is composed of two proteolyzed inhibitor subunits linked to one thrombin molecule. This intersubunit link maintains the integrity of the α₂M in sodium dodecyl sulfate, accounting for the high molecular weight bands under nonreducing conditions. Comparison with a synthetically cross-linked α₂M molecule allows a tentative but not unambiguous assignment of one of the bands to this novel structure.