Binding site of a dextran-specific homogeneous IgM: thermodynamic and spectroscopic mapping by dansylated oligosaccharides

Abstract

The hapten binding properties of the homogeneous mouse Ig[immunoglobulin]M secreted by MOPC-104E [plasmacytoma cells] were investigated. Hapten-association constants were determined by equilibrium and displacement equilibrium dialysis or by fluorometric titrations of the protein with the fluorescent derivatives of the haptens. For the latter type of measurements, several oligosaccharides were derivatized to the corresponding dansylhydrazones. The synthesis, generally applicable to oligosaccharides with free reducing ends, is described. Analysis of the thermodynamic parameters for the binding of 18 haptens forms the basis for proposing a model of the binding site of MOPC-104E. This model is supported and refined by results of the measurements of linear and circular polarization of the fluorescence of the dansylated haptens. The binding site apparently consists of a cavity with about 12-.ANG. depth, complementary to a terminal nonreducing nigerosyl group. At the entrance to this cavity, a further subsite is identified forming interactions of lower specificity with an additional glucose unit.