Methanethiolation of the Liberated Cysteine Residues of Human α2-Macroglobulin Treated with Methylamine Generates a Derivative with Similar Functional Characteristics as Native α2-Macroglobulin

Abstract

The thiol-modifying reagent methyl methanethiosulfonate reacts with the cysteine residues of thiol esters released upon treatment of human α2-macroglobulin with methylamine. This methanethiolation generates a derivative of α2-macroglobulin, with an ‘open trap’ and slow mobility in non-denaturing PAGE, similar to native α2-macroglobulin. This similarity is further substantiated by surface hydrophobicity determinations and by the fact that neither the derivative nor native α2-macroglobulin are cleared from the circulation in mice. Cleavages of bait regions in the derivative and native α2-macroglobulin, however, result in electrophoretically fast forms which are cleared from the circulation in mice. In contrast to native α2-macroglobulin, which can bind 2 mol chymotrypsin/mol, α2-macroglobulin treated with methylamine and methylmethanethiosulfonate binds only 0.8 mol chymotrypsin/mol. Protection of trypsin against inhibition by soybean trypsin inhibitor is significantly better when α2-macroglobulin is modified by methylamine and methylmethanethiosulfonate than when it is modified by dinitrophenyl thiocyanate, which cyanylates the exposed thiol group. The methanethiolated derivative is also more stable than the corresponding cyanylated derivative in that it is transformed to an electrophoretically fast form with a half-life of 9 h as compared to a half-life of 7 h for the latter. The transformation to the fast form is not due to instability of the thiol modification.