Thrombin-induced conformational changes of human .alpha.2-macroglobulin: evidence for two functional domains

Abstract

The interaction of thrombin with .alpha.2-macroglobulin (.alpha.2M) was characterized by monitoring conformational changes and measuring the increase of free sulfhydryl groups during the reaction. Under experimental conditions where [thrombin] > [.alpha.2M], the conformational change, measured by increases in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate and thiol group appearance displayed biphasic kinetics. The initial rapid phase results in the formation of a stable complex, the appearance of 2 sulfhydryl groups, the cleavage of approximately half of the MW 180,000 subunits and a conformational change that is not as extensive as that which occurs with trypsin. The slower phase is associated with the appearance of 2 additional sulfhydryl groups, increased cleavage of the MW 180,000 subunit and additional conformational changes. The slow phase apparently results from hydrolysis of the MW 180,000 subunit(s) due to proteolysis of the .alpha.2M-thrombin complex by free thrombin. Experiments with 125I-thrombin document the binding of 1 mol of thrombin/mol of .alpha.2M that is not dissociated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex. At higher rates of thrombin to .alpha.2M, a 2nd mole of thrombin will reversibly associate with the 1:1 .alpha.2M-thrombin complex. Under conditions where [thrombin] < [.alpha.2M], biphasic kinetics were not observed, and the conformational change, sulfhydryl appearance and hydrolysis of the MW 180,000 subunit were found to follow 2nd-order kinetics. The 2nd-order rate constants obtained for these events were (3.2 .+-. 0.5) .times. 103 M-1 s-1, (2.5 .+-. 0.4) .times. 103 M-1 s-1, and (4.9 .+-. 0.7) .times. 103 M-1 s-1, respectively. The similarity of these rate constants is consistent with a concreted mechanism or with a sequential mechanism in which either thrombin association or cleavage of the .alpha.2M subunit(s) is rate limiting and the subsequent events (i.e., thiol ester hydrolysis and conformational change) occur at more rapid rates.