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 centre, and this may be suggestive evidence for a reactive alpha 2M centre 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 further 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.
R D
RD
Feinman
A I
AI
Yuan
S R
SR
Windwer
D
D
Wang
D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D000511:alpha-Macroglobulins / Q000378:metabolism*
D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; D013917:Thrombin