KINETICS OF MULTIVALENT ANTIGEN DNP-BSA BINDING TO IGE-FC-EPSILON-RI IN RELATIONSHIP TO THE STIMULATED TYROSINE PHOSPHORYLATION OF FC-EPSILON-RI

Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE bound to Fc epsilon RI to stimulate cellular responses, although key feature s of the binding process are unknown. Fluorescence quenching can be us ed to study the kinetics of DNP-BSA binding to FITC-IgE. We observe th at DNP-BSA binds more slowly to IgE than does an equimolar amount of a monovalent DNP ligand, suggesting that the average effective number o f DNP groups per BSA is less than one, The binding data are well descr ibed by a transient hapten exposure model in which most of the DNP gro ups are unavailable for binding but have some probability of becoming exposed and available for binding during the time of the binding measu rement. Additional experiments indicate that, for suboptimal to optima l concentrations of DNP-BSA, most of the FITC fluorescence quenching o n the cell surface is due to cross-linking events. With these concentr ations at 15 degrees C, the kinetics of FITC fluorescence quenching by DNP-BSA correlates with the kinetics of DNP-BSA-stimulated tyrosine p hosphorylation of Fc epsilon RI. At 35 degrees C, the phosphorylation kinetics are biphasic during the time period in which cross-linking co ntinues to increase, Our results establish a quantitative relationship between the timecourse for cross-linking by multivalent Ag and Fc eps ilon RI-mediated signaling, and they provide the means to predict the kinetics of cross-linking under a wide variety of conditions.