Binding of phosphorylcholine to an IgM Waldenström as studied by fluorescence spectroscopy and circular dichroism.
The binding characteristics of an IgM Waldenström(FR) for the ligand phosphorylcholine has been 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 maximun (5nm). The same properties were observed with the 7S IgM subunits. The association constant KA for phosphorylcholine was 6X10(4) M-1 FOR IgM FR and the 7S subunit, as determined by fluorescence titration, a value in agreement with the 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. In contrast, only 4% of fluorescence enhancement was noted upon binding in 3 M GuHC1 and no enhancement could be seen when the concentration of  GuHC1 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 and 8, whereas 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 fluorescnece 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
D000818:Animals; D002794:Choline / Q000031:analogs & derivatives; D002942:Circular Dichroism; D006146:Guanidines; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007075:Immunoglobulin M; D051379:Mice; D009194:Myeloma Proteins; D010767:Phosphorylcholine; D011485:Protein Binding; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014508:Urea; D008258:Waldenstrom Macroglobulinemia / Q000097:blood*
D006146:Guanidines; D007075:Immunoglobulin M; D009194:Myeloma Proteins; D010767:Phosphorylcholine; D014364:Tryptophan; D014508:Urea; D002794:Choline