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., & 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 α2-macroglobulin (α2M) normally resulting from reaction of α2M with methylamine. The kinetics of reaction of DNPSCN with α2M in the presence of methylamine are examined here and shown to approximate pseudo first order, reflecting the rate-limiting reaction of α2M with methylamine [Larsson, L. J., & Björk, I. (1984) Biochemistry 23, 2802-2807]. One mole of DNPS− is liberated per mole of free thiol in α2M, 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 α2M 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 α2M from the “slow” to the “fast” electrophoretic form. Furthermore, DNPSCN added after the conformational change can partially reverse the change. A similar reversal can be effected by cyanylation, with NaCN, of methylamine-treated α2M 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 α2M. 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 α2M and point to a crucial role for the thiol groups. © 1990, American Chemical Society. All rights reserved.