Acquisition of the covalent quaternary structure of an immunoglobulin G molecule. Reoxidative assembly in vitro

Abstract

Results were recently reported of an investigation of the reoxidation of a human, monoclonal immunoglobulin G, following selective reduction of its interchain disulfides by dithiothreitol. In that work, the reoxidative behavior of the molecule under nondissociating conditions was described. In the present paper, results are presented of the reoxidation of H and L chains of this protein alone, or mixed in varying proportions after separation, or mixed with the L chains modified prior to recombination and reoxidation. The overall reoxidative assembly patterns in experiments with H and L separated prior to recombinations are similar to those observed when the chains remain noncovalently associated throughout. With equimolar mixtures of H and L, the reoxidation rates also are similar to those of unseparated chains. When L chains are present in excess, the overall in vitro rates of covalent assembly are generally diminished, probably indicating transient nonproductive interactions. At the highest molar excesses of L (3:1), the assembly pathways may also be modified. In all experiments with excess L chains, covalent L2 dimers form at rats which are comparatively slow relative to the H2L2 assembly rates. Two kinds of reoxidation experiments with modified L chains are described here for the 1st time. In the first, the free half-cystine of L is irreversibly blocked by reaction with iodoacetamide, and the alkylated L chains are recombined with reduced H chains. This experiment isolates the reactions in which H2 disulfides are formed without the accompanying formation of HL bonds. Although the alkylated L chains do not play a direct role in the reoxidation, their presence is required to inhibit aggregation and precipitation of high MW products which otherwise ensue; this suggests a possible biological role for excess L in vivo. In the 2nd kind of experiment, covalent L2 dimers are mixed with reduced H chains. L2 rapidly disappears with the concurrent appearance of HL, H2L, and fully assembled H2L2. H2 dimers are also reactive in this process. Special procedures were developed for analyzing the data from these experiments. A complete format is given for the quantitative determination of the concentration of each of the molecular components directly from spectroscopic scans of the gels. The computational methods solve the general analytical problem posed when staining is not proportional to mass and are applicable to a wide variety of systems utilizing gel electrophoresis to study subunit interactions.