Dissociability of enzyme-α₂-macroglobulin complexes

Experiments were performed to measure the extent to which enzymes bound to α₂macroglobulin (α₂M) could be dissociated from the complex. Noncovalent complexes are known to exist between α₂M and proteases, such as methyl-trypsin that have had their lysyl amino covalently blocked. Complexes between the inhibitor and native enzymes also have a certain fraction noncovalent binding. Because of the severe steric hindrance imposed on enzymes bound to α₂M, even in the noncovalent mode, it has been proposed in the literature that they are not dissociable in the usual sense but, rather, are "trapped" in clathrate-like complexes. The results presented here show that lysyl-blocked methyl-thrombin, or native thrombin are released from their α₂M complex by an excess of other lysyl-blocked or native proteases. Under conditions where native thrombin is displaced, labeled enzymes can be incorporated, indicating the inhibitor is intact by the criterion of incorporating enzymes. Likewise, native elastase can be released from its α₂M complex by excess cold elastase or the inactive anhydrotrypsin, the latter experiment being carried out with an excess of the low-molecular-weight inhibitor diisopropyl phosphofluoridate. In conjunction with previous results showing that lysyl-blocked enzymes are removed from α₂M by soybean trypsin inhibitor, the data indicate that, however sterically hindered, α₂M-bound enzymes are dissociable and no unique "trapped" intermediate need be postulated.