Inhibition and partial reversal of the methylamine-induced conversion of "slow" to "fast" electrophoretic forms of human .alpha.2-macroglobulin by modification of the thiols
It has been shown previously [Van Leuven, F., Marynen, P., Cassiman, J.J., and Van den Berghe, H. (1982) Biochem. J. 203, 405-411] that 2,4-dinitrophenyl thiocyanate (DNPSCN) can block the conversion of "slow" to "fast" electrophoretic forms of human .alpha.2-macroglobulin (.alpha.M) normally resulting from reaction of .alpha.2M with methylamine. The kinetics of reaction of DNPSCN with .alpha.2M in the presence of methylamine are examined here and shown to approximate pseudo first order, reflecting the rate-limiting reaction of .alpha.2M with methylamine [Larsson, L. J., and Bjork, I. (1984) Biochemistry 23, 2802-2807]. One mole of DNPS- is liberated per mole of free thiol in .alpha.2M, consistent with cyanylation of the thiol liberated upon scission of the internal thiol esters by methylamine. I3- can also react with the methylamine-generated thiol groups of .alpha.2M with a stoichiometry consistent with conversion of the thiol to a sulfenyl iodide. Reaction of the thiol groups with either DNPSCN or I3- inhibits the conversion of .alpha.2M from the "slow" to the "fast" electrophoretic form. Furthermore, DNPSCN added after the confrontal change can partially reverse the change. A similar reversal can be effected by cyanylation, with NaCN, of methylamine-treated .alpha.2M in which the liberated thiols have first been converted to mixed disulfides by reaction with dithiobis(nitrobenzoic acid). Differential scanning calorimetry shows nearly identical properties for the methylamine-treated "fast" form and the cyanylated "slow" form of .alpha.2M. In contrast, the susceptibility to dissociation by urea or mercaptoethanol of the cyanylated "slow" form is nearly the same as that of the native "slow" form and very different from that of the "fast" form. These properties, together with the ready blocking or reversal of conformational change by cyanylation, highlight the small energy differences that appear to be involved in the domain rearrangement of .alpha.2M and point to a crucial role for the thiol groups.