Binding of phosphorylcholine to an IgM Waldenstroem as studied by fluorescence spectroscopy and circular dichroism

Abstract

The binding characteristics of a [human] IgM [immunoglobulin M] Waldenstrom (FR) for the ligand phosphorylcholine was studied by fluorescence spectroscopy. Upon phosphorylcholine addition, IgM FR exhibited 83% enhancement of the tryptophanyl fluorescence which was associated with a red shift of the emission maximum (5 nm). The same properties were observed with the 7S IgM subunits. The association constant KA for phosphorylcholine was 6 .times. 104 M-1 for IgM FR and the 7S subunit as determined by fluorescence titration a value in agreement with that obtained by equilibrium dialysis. No significant decrease in the KA value was found in the presence of 3 M urea; in 6 M urea, the increase in fluorescence intensity was 36% of the value obtained in the absence of denaturing agent. Only 4% of fluorescence enhancement was noted upon binding in 3 M GuHCl [guanidine hydrochloride] and no enhancement could be seen when the concentration of GuHCl was increased to 5 M, thus suggesting complete unfolding of the protein and subsequent loss of binding activity. The pH dependence study of the phosphorylcholine binding to IgM FR indicated no significant differences in the fluorescence enhancement between pH 5-8; at more acidic or alkaline pH values, the enhancement became smaller. At pH 3.0 and 10.0, no enhancement was seen, suggesting no binding of the ligand, a fact confirmed independently by equilibrium dialysis. When the spectroscopic properties of the IgM FR were compared with those of murine myeloma proteins that bind the same ligand, large differences were recorded in the amplitude of the phosphorylcholine induced enhancement of the fluorescence and in the shift of the emission maximum wavelength. This suggests that the human and murine proteins interact differently with the small ligand phosphorylcholine thus implying that the variable domains of these molecules are not identical.