Molecular basis for the temperature-dependent insolubility of cryoglobulins. X. The amino acid sequence of the heavy chain variable region of McE.

The amino acid sequence of the VH region of McE, a monoclonal IgM cryoimmunoglobulin, has been determined employing automated sequencing methodology. Digestion of the intact Fab mu component, derived by trypsin cleavage of the parent protein at elevated temperature with CNBr, followed by complete reduction and alkylation, yielded the intact light chain as well as the 2 CNBr fragments that constituted the VH. N-terminal sequencing of the larger unblocked CNBr fragment, along with a component fragment derived by cleavage by BNPS-Skatole, established the structure of the VH from position 88 through the V leads to C switch region. Citraconylation of the smaller, blocked fragment effected sufficient solubilization for enzymatic deblocking and direct sequencing of the N-terminal 20 residues of the VH. Complete trypsin digestion of the N-terminal CNBr fragment yielded 9 peptides that could be isolated by preparative cation exchange chromatography and gel filtration. The complete sequence of these peptides along with 4 chymotryptic peptides completed the primary structure of the VH region. The primary structure of McE appears to resemble that of He, previously identified as belonging to the VH II subgroup. The presence of characteristic CDR and FR regions as well as the identification of a probable site of glycosylation suggest that the cryoimmunoglobulin closely resembles noncryoglobulins in terms of overall structural composition. The cryoglobulin property may arise through alterations in individual residues or unfavorable arrangements of CDR and FR segments.

Covalent immunoglobulin assembly in vitro: reactivity of light chain covalent dimers (L2) and blocked light chain monomers.

Covalent light chain dimers (L2) and cysteine-blocked L chain monomers readily react with partially reduced heavy (H) chains. A rapid disappearance of these blocked L chain species is followed by the appearance of covalent intermediates-HL, H2, and H2L-leading to fully assembled H2L2. The mechanism of initial disulfide bond formation between heavy and light chains is disulfide interchange.

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)).