Electrophoretic and spectroscopic analyses of equine alpha 2-macroglobulin with cleavage of the thiol ester bonds by methylamine.
Reaction of equine alpha 2-macroglobulin (alpha 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 alpha 2M inhibited two molecules of trypsin, similar to human alpha 2M. The methylamine-treated equine alpha 2M, with complete cleavage of the thiol ester bonds, still inhibited the activity of trypsin, though human alpha 2M lost its inhibitory activity by treatment with methylamine. These results indicate that the mode of inhibition of trypsin by equine alpha 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 alpha 2M to bind the enzyme. Ultraviolet absorption difference, intrinsic fluorescence, and circular dichroism spectra of the methylamine-treated equine alpha 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 alpha 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 alpha 2M when each had reacted with trypsin.
D000818:Animals; D002942:Circular Dichroism; D006736:Horses / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D000511:alpha-Macroglobulins / Q000378:metabolism*
D008744:Methylamines; D000511:alpha-Macroglobulins; C027451:methylamine