Binding mechanisms of PEGylated ligands reveal multiple effects of the PEG scaffold.
A series of synthetic ligands consisting of poly(ethylene glycol) (PEG), capped on one or both ends with the hapten 2,4-dinitrophenyl (DNP), were previously shown to be potent inhibitors of cellular activation in RBL mast cells stimulated by a multivalent antigen [Baird, E. J., Holowka, D., Coates, G. W., and Baird, B. (2003) Biochemistry 42, 12739-12748]. In this study, we systematically investigated the effect of increasing length of the PEG scaffold on the binding of these monovalent and bivalent ligands to anti-DNP IgE in solution. Our analysis reveals evidence for an energetically favorable interaction between two monovalent ligands bound to the same receptor, when the PEG molecular mass exceeds approximately 5 kDa. Additionally, for ligands with much higher molecular masses (>10 kDa PEG), the binding of a single ligand apparently leads to a steric exclusion of the second binding site by the bulky PEG scaffold. These results are further corroborated by data from an alternate fluorescence-based assay that we developed to quantify the capacity of these ligands to displace a small hapten bound to IgE. This new assay monitors the displacement of a small, receptor-bound hapten by a competitive monovalent ligand and thus quantifies the competitive inhibition offered by a monovalent ligand. We also show that, for bivalent ligands, inhibitory capacity is correlated with the capacity to form effective intramolecular cross-links with IgE.
D019297:2,4-Dinitrophenol / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000465:Algorithms; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D007073:Immunoglobulin E / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008024:Ligands; D008956:Models, Chemical; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D008024:Ligands; D007073:Immunoglobulin E; D011092:Polyethylene Glycols; D016650:Fluorescein-5-isothiocyanate; D019297:2,4-Dinitrophenol