ELECTROPHORETICALLY SLOW AND FAST FORMS OF THE ALPHA-2-MACROGLOBULIN MOLECULE

α(2)-Macroglobulin (α(2)M) was isolated from human plasma by a four-step procedure: poly(ethylene glycol) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No cantaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-α(2)M. S-{2)M bound about 2mol of trypsin/mol. Treatment of S-α(2)M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-α(2)M). The electrophoretic mobility of the F-α(2)M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the α(2)M to a conformational change, but there was no evidence of a change in pI or Stokes radius. Electrophoresis of S-α(2)M in the presence of sodium dodecyl sulphate gave results consistent with the view that the α(2)M molecule is a tetramer of identical subunits assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thirds-length and one-third-length chains, however. This was not apparent with F-α(2)M produced by ammonium salts. F-α(2)M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-α(2)M gave 'rockets' 12-29% lower than those with S-α(2)M. The nature of the interactions between subunits in S-α(2)M and F-α(2)M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-α(2)M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-α(2)M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact α(2)M. F-α(2)M was less easily dissociated than was S-α(2)M. S-α(2)M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Intact reactive α(2)M could then be regenerated in high yield by reoxidation of the subunits. F-α(2)M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-α(2)M were blocked by carboxylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-)tetramers. It is emphasized that F-α(2)M does not have the properties of a denatured form of the protein. The suggestion is made that one attractive interpretation of the difference in properties between the S- and F-forms is that the S-form is a planar tetramer, whereas the F-form is pseudotetrahedral. The results are considered in relation to the 'trap' hypothesis for the reaction of α(2)M with proteinases.