IgE antibodies cause long-term sensitization of tissue mast cells and blood basophils toward allergen-induced cross-linking and triggering of allergic inflammation. This persistence of IgE binding is due to its uniquely high affinity for the receptor FcepsilonRI and in particular its slow rate of dissociation once bound. The binding interface consists of two subsites, one contributed by each Cepsilon3 domain of IgE Fc in a 1:1 complex. We have investigated the contributions of Cepsilon3 disulfide linkage and glycosylation to the kinetics and affinity of binding of an Fc subfragment (Fcepsilon3-4) to a soluble receptor fragment (sFcepsilonRIalpha). In contrast to IgG Fc where deglycosylation abrogates receptor binding activity, the removal of the N-linked carbohydrate at Asn-394 in Fcepsilon3-4 only reduces binding affinity by a factor of 4, principally because of a faster off-rate. Removal of the inter-heavy chain disulfide bond unexpectedly resulted in a fragment with a much faster off-rate and the potential to form a complex with a 2:1 stoichiometry (sFcepsilonRIalpha:Fcepsilon3-4). This permitted the determination of the affinity of a single, natively folded Cepsilon3 domain for the first time. The low affinity Ka approximately 10(5)-10(6) m-1, similar to that determined previously for an isolated and partially folded Cepsilon3 domain, demonstrates that substantial reduction in affinity can be achieved by preventing the engagement of one of the two Cepsilon3 domains. Recent structural data indicate that conformational change in IgE is required to allow both Cepsilon3 domains to bind, and thus an allosteric inhibitor that prevents access to the second Cepsilon3 has the potential to reduce the ability of IgE to sensitize allergic effector cells.