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

We recently reported results of an investigation of the reoxidation of a human, monoclonal immunoglobulin G, following selective reduction of its interchain disulfides by dithiothreitol (Sears, D.W., et al. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 353). In that work, we described the reoxidative behavior of the molecule under nondissociating conditions. In the present paper, results are presented of the reoxidation of heavy (H) and light (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 asembly patterns in experiments with H and L separated prior to recombination 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. However, 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 rates which are comparatively slow relative to the H2L2 assembly rates. Two kinds of reoxidation experiments with modified L chains are described here for the first 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-molecular-weight products which otherwise ensue; this suggests a possible biological role for excess L in vivo. In the second 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 H2L. 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. A theoretical analysis of pathway and kinetic cooperatively in this system is presented in the following paper (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (following paper in this issue)).

Relative susceptibilities of the interchain disulfides of an immunoglobulin G molecule to reduction by dithiothreitol.

The reduction by dithiothreitol (DTT) of the four interchain disulfides of a human IgGlkappa immunoglobulin has been studied by two methods: variation of the concentration of DTT relative to the protein concentration (incremental reduction); and variation of the time of reduction at fixed levels of DTT and protein (kinetic reduction). In both cases, the results depend on whether the reduction is carried out aerobically or anaerobically. Under aerobic conditions, the relative levels of intermediates (HL, H2, and H2L) which are generated as native molecules (H2L2) are converted to reduced heavy (H) and light (L) chains depend on the concentrations of protein and DTT as well as on the exposure time to DTT; no stable equilibrium is reached between reduced and oxidized states and conditions gradually revert from those favoring reduction to those favoring reoxidation. By contrast, anaerobic reduction is independent of protein concentration or time of exposure to DTT, beyond about 30 min, indicating that an equilibrium between partially reduced and oxidized states is achieved. The distribution of intermediates observed under anaerobic conditions has been analyzed according to theoretical models (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in a series of three articles in this issue)). Within experimental error, both kinds of anaerobic experiments resemble a random reduction process wherein the four disulfides are equivalent and independent of each other with respect to rate and extent of reduction by D. It is concluded that there are no readily detected pathways in the process, as would occur if the intrinsic reactivities of the bonds were distinct, and no marked cooperatively between the four reaction sites, as would be observed if reduction of one bond materially facilitated or hampered reactivity at another site. Both of these characteristics of the reduction are in direct contrast to those of the reoxidative process, which is marked by the initial preference for formation of a bond between heavy and light chains, and by kinetic cooperativity in bond formation during the course of the reaction (Sears, D.W., et al. (1977), Biochemistry 16 (first in a series of three articles in this issue); Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in this series)).

A kinetic study in vitro of the reoxidation of interchain disulfide bonds in a human immunoglobulin IgGLk. Correlation between sulfhydryl disappearance and intermediates in covalent assembly of H2L2.

After reduction by dithiothreitol and removal of the reductant by molecular sieve chromatography, the four interchain disulfide bonds of the human IgGlk protein Fro reoxidize in the presence of oxygen and trace metal ions. The six molecular components of the reoxidation--L (light chain), H (heavy chain), HL, H2, H2L, H2L2--are quantitatively determined from polyacrylamide gels containing sodium dodecyl sulfate and the time-dependent sulfhydryl titer is measured with 5,5'-dithiobis-(2-nitrobenzoic acid). The rates of H2L2 covalent assembly depend on pH in an unexpected way: If the reduced protein is chromatographed at pH 3.2 and then adjusted to pH 7.5 (25 degrees, ionic strength equals 0.14), H2L2 formation proceeds rapidly, with half-times ranging between 20 and 40 min. In contrast, if chromatography is carried out at pH 5.5 before adjusting to the same final conditions, the half-times for H2L2 formation are considerably longer (120-180 min). The half-times in the former case approach the somewhat faster rates of H2L2 assembly observed in pulse-chase experiments with various types of mouse, IgG-producing cells [Baumal, et al. (1971) J. Exp. Med. 134, 1316-1334]. To facilitate comparison of experiments and models, we plot the concentrations of the six components against the corresponding number of sulfhydryl equivalents per mole of Fro. The respective plots for the pH 3.2 leads to 7.5 and 5.5 leads to 7.5 experiments are very similar despite the rate differences. Moreover, these plots differ significantly from the calculated plot for a hypothetical random reoxidation in which the intrinsic probability for formation of each correct HL and H2 disulfide bond is assumed equal and independent. It is concluded that the in vitro reoxidation of Fro (i) is other than random; (ii) involved a pathway of pathways with HL, H2, and H2L precursors; and (iii) involves at least some kinetic cooperativity in bond formation, since no model bases solely on independent bond formation adequately accounts for the results. The models were used also to examine the cellular assembly pathways of mouse IgG proteins.