Covalent assembly of mouse immunoglobulin G subclasses in vitro: application of a theoretical model for interchain disulfide bond formation

The pathways and kinetics of interchain disulfide bond formation have been determined in vitro for purified myeloma proteins representing the three major subclasses of mouse immunoglobulin G (IgG)using the reoxidation system described previously (Petersen, J. G. L. &Dorrington, K. J. (1974) J. Biol. Chem. 249, 5633–5641). Mixtures of oxidized and reduced glutathione were added to act as a disulfide interchange catalyst. The pathways of covalent assembly observed in vitro were qualitatively and quantitatively similar to those followed by the various subclasses in vivo. HH and HHL were the principle covalent intermediates seen with IgG1 (MOPC 31C) and IgG2a (MOPC 173 and clone 19). With IgG2b (MPC 11C), HL, HH and HHL were all prominant intermediates.The time courses of reoxidation were simulated using a theoretical model based on second-order reaction kinetics (Percy, J. R., Percy, M. E. &Dorrington, K. J. (1974) J. Biol. Chem. 250, 2398–2400). Two distinct phases were apparent in the reoxidation sequence. The first, which lasted for the initial 5–15 min, did not conform to the theoretical model. The second phase could be accounted for by the model and represented the remainder of the covalent assembly process. The physico-chemical basis for this biphasic phenomenon was explored. Sedimentation velocity studies showed that noncovalent association was incomplete at the beginning of the reoxidation step for all proteins except IgG2b (MOPC 11C). No dissociation was apparent in the reduced and alkylated proteins at pH 5 in the absence of prior exposure to acid conditions. Thus, exposure to acid appears to affect the affinity between the subunits in the native proteins. Transfer of the proteins from pH 5 to pH 8.2 (the pH at which reoxidation proceeds) is accompanied by the generation of an absorption difference spectrum over an 8–10-min period. These data suggest that a pH-dependent conformational relaxation process may influence the early stages of reoxidation.