Kinetics of appearance of sulfhydryl groups in alpha 2-macroglobulin on reaction of the inhibitor with amines.

The mechanism of the appearance of sulfhydryl groups in alpha 2-macroglobulin in the reaction with amines was characterized by analyses of the kinetics with ammonia and methylamine. All reactions occurred under pseudo-first-order conditions in the range of pH (7.0-8.6) and amine concentration (10-600 mM) investigated. The logarithm of the pseudo-first-order rate constant increased linearly as a function of pH with a slope of unity, indicating that the unprotonated amine is the active species in the reaction. Plots of the observed pseudo-first-order rate constants vs. concentration of unprotonated amine at constant pH were also linear and gave second-order-rate constants of 0.32 and 13.8 M-1 s-1 for ammonia and methylamine, respectively, at pH 8.0; similar values were obtained at pH 8.6. Activation energies of 85 and 100 kJ mol-1 and activation entropies of 10 and 95 J K-1 mol-1 for ammonia and methylamine, respectively, were estimated from Arrhenius plots, suggesting that the higher reaction rate for methylamine is due primarily to a higher activation entropy. These results are consistent with the release of sulfhydryl groups being caused by a nucleophilic attack of the uncharged amine on a thio ester bond of alpha 2-macroglobulin in a bimolecular reaction occurring under pseudo-first-order conditions. The characteristics of the reaction suggest that the thio ester in each alpha 2-macroglobulin subunit reacts independently and equivalently with the amine and also that the thio ester bond cleavage initiates the reaction sequence leading to inactivation of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)