Electrophoretic and spectroscopic analyses of equine α 2-macroglobulin with cleavage of the thiol ester bonds by methylamine

Abstract

Reaction of equine α 2-macroglobulin (α 2M) with methylamine caused generation of 3.7 mol of thiol groups per mole of the protein, and the second-order rate constant of the generation was calculated to be 3.5 m −1 s −1. The inhibitory profile of caseinolytic activity of trypsin indicated that one molecule of equine α 2M inhibited two molecules of trypsin, similar to human α 2M. The methylamine-treated equine α 2M, with complete cleavage of the thiol ester bonds, still inhibited the activity of trypsin, though human α 2M lost its inhibitory activity by treatment with methylamine. These results indicate that the mode of inhibition of trypsin by equine α 2M is substantially unperturbed by cleavage of the thiol ester bonds and that the intact thiol ester bonds per se are not essential for the ability of equine α 2M to bind the enzyme. Ultraviolet absorption difference, intrinsic fluorescence, and circular dichroism spectra of the methylamine-treated equine α 2M showed that this treatment caused only a small change in conformation of the protein. Reaction of the methylamine-treated protein with trypsin induced appreciable changes in the spectra, indicating a large change in conformation of the protein. These findings were consistent with the results obtained by electrophoresis: The band of methylamine-treated equine α 2M showed indistinguishable mobility from that of the unmodified protein, indicating that no appreciable change in conformation occurred, and distinctly different mobility from that of the unmodified or methylamine-treated equine α 2M when each had reacted with trypsin.