A General Method for the Isolation of the VH Domain from IgM and Other Immunoglobulins

Abstract

A chemical cleavage procedure has been developed for IgM, which leads to the isolation of a fragment (Vµ) consisting of the V_H region and a small number of N-terminal residues from the Cµ1 domain. The sulfhydryl groups resulting from the reduction of the L-H disulfide linkage are converted to the S-cyano form by treatment with 2-nitro-5-thiocyanobenzoic acid. Cleavage of the H chain on the amino side of the cyanylated residue is effected by incubation overnight at pH 9 and 37°C. The Vµ fragment is the only one-domain fragment formed and is separated from the other products by gel chromatography with Sephacryl S-200 in 6 M guanidine-HCl. The Vµ fragment has been identified by its C-terminal residues on the basis of the established sequence of the human µ-chain. Additional characterization and verification was provided by isoelectric focusing and gel chromatography. The cleavage procedure is probably generally applicable to IgM of all species. This conclusion is based on the anticipation that the decisive structural feature of the disulfide linkage of the L chain to a half-cystine located near the N-terminal end of the Cµ1 domain is a general feature of IgM structure. Since this arrangement of the L-H bond also is found in other classes and subclasses of immunoglobulin isolation of V_H-containing fragments is probably feasible in these instances. Among the many uses of Vµ (and other similar V_H-containing fragments) are the preparation of anti-idiotype antibody specific for V_H determinants, the evaluation of the contribution of V_H to the binding of ligand, and the identification of the V_H domain on B and T lymphocytes with antisera specific for the framework determinants of V_H.