Ligand binding and physicochemical characteristics of an IgM mouse plasmacytoma ABPC-22

Immunoglobulin IgM₂₂, secreted by mouse plasmacytoma ABPC-22, was characterized on the basis of ligand binding and physicochemical properties. Upon purification, only two polypeptides (μ aqnd κ chains) were detected by 6 M urea isoelectric focusing in polyacrylamide and SDS-polyacrylamide electrophoresis (SDS-PAGE). The μ and κ chains possessed pI values of 6.27 and 6.13 respectively, in 6 M urea. The molecular masses of μ and κ chains were established by SDS-PAGE and molecular sieve chromatography to be 70 and 30 kd, respectively. Thus, a molecular weight of 1 × 10⁶ was estimated for non-reduced IgM₂₂, and was confirmed by molecular sieve chromatography and sedimentation equilibrium ultracentrifugation. J chain was detected in freshly purified samples. As shown by competitive hapten inhibition (CHI) of the precipitation assay, using ¹²⁵I-rhodamine B₅BSA as the reference ligand, IgM₂₂ bound a wide range of structurally related and unrelated probes. Competing for the same site were polyaromatic anions (such as pyrene-1-sulfonate, rhodamine B and fluorescein) and weakly inhibitory organic acids (such as acetate and propionate). In addition. Fab fragments inhibited precipitation of ¹²⁵I-rhodamine B₅BSA by IgM₂₂. Equilibrium dialysis studies revealed a K_a f 1.5 × 10³ M^-1 for the interaction of ¹⁴C-fluorescein with IgM₂₂. A K_a of 3.2 × M^-1 for rhodamine B was determined using a molecular sieve column equilibrated in varying concentrations of rhodamine B. Based on relative K_a values, CHI results and fluorescence binding data with anilinonaphthalene sulfonate (ANS), the nature of the antibody active site was described in detail.