An in Vitro System for Studying the Kinetics of Interchain Disulfide Bond Formation in Immunoglobulin G

Abstract An in vitro system for studying the order and kinetics of interchain disulfide bond formation (covalent assembly) in immunoglobulins has been developed using subclasses 1 and 4 of human immunoglobulin G. Noncovalent association of reduced, but not alkylated, heavy and light chains was achieved at pH 4.8 and air oxidation initiated by rapid transfer to aerated buffer at pH 8.2. Disulfide-bonded species in samples taken at various times were detected, following alkylation, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The principal intermediates in the covalent assembly of immunoglobulin G1, with either kappa or lambda light chains, were half-molecules (HL) and H2L. The half-time for the appearance of the completed molecule, H2L2, ranged from 70 to 95 min at 25°. Half-molecules were the predominant intermediate in the assembly of immunoglobulin G4. The disulfide bonds between the heavy chains formed slowly; H2L2 appeared with a half-time of 16 hours. The effects of temperature, pH, cupric ion concentration, and reducing agent on the air oxidation of reduced immunoglobulin were evaluated. Although all these parameters influenced the rate of H2L2 production, the order of disulfide bond formation was unaffected except in the presence of high concentrations of cupric ion. Covalent assembly utilizing a thiol-disulfide interchange system (oxidized and reduced glutathione) was also examined. Optimum rates of H2L2 production were observed with 10-2 m glutathione + 10-3 m oxidized glutathione. For immunoglobulin G1 assembly the half-time was reduced to 25 min at 25° and 10 min at 37° and to 30 min at 37° for subclass 4. These rates are comparable to those observed for covalent assembly in vivo.