KINETICS OF THE REACTION OF THROMBIN AND ALPHA-2-MACROGLOBULIN

The kinetics of the reaction of .alpha.2-macroglobulin (.alpha.2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native .alpha.2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester center, and this may be suggestive evidence for a reactive .alpha.2M center that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several .alpha.2M chains by the bound proteinase, providing furthe evidence that the very-high-Mr species seen on gels may arise from dimers of the .alpha.2M molecule held together by covalent bonds to the enzyme.