THE FC SEGMENT OF IGE INFLUENCES THE KINETICS OF DISSOCIATION OF A SYMMETRICAL BIVALENT LIGAND FROM CYCLIC DIMERIC COMPLEXES

As part of a systematic effort to determine the features of immunoglob ulin E-receptor (IgE-Fc epsilon RI) aggregation that are critical for cellular activation, we used fluorescence to examine the dissociation of a soluble bivalent ligand, -dinitrophenyl)amino]caproyl]-L-tyrosyl] -L-cystine ((DCT)(2)-cys), from soluble bivalent IgE and its bivalent F(ab')(2) and monovalent Fab' fragments. Cross-linking of Fab' fragmen ts by (DCT)(2)-cys is limited to linear dimers, and we find that (DCT) (2)-cys dissociation from Fab' occurs with a single kinetic coefficien t [(4.2 +/- 0.6) x 10(-3) s(-1)] that corresponds to the lower of the two kinetic coefficients observed with the bivalent IgE [(4.7 +/- 0.7) x 10(-2) s(-1) and (4.4 +/- 0.3) x 10(-3) s(-1)]. Similarly, the lowe r value is obtained for dissociation of (DCT)(2)-cys that is monovalen tly bound to IgE after incubation with a large excess of the ligand. ( DCT)(2)-cys can bind to bivalent F(ab')(2) fragments and form a variet y of linear and cyclic aggregates, similarly to IgE, but, unlike IgE, we find that dissociation occurs with a single kinetic coefficient sim ilar to that observed for Fab'. We find that IgE and its (Fab')(2) fra gments form highly stable cyclic dimer rings with two (DCT)(2)-cys. We demonstrate that the kinetic coefficients are independent of enhanced fluorescence quenching observed for bound sites in cyclic dimers. Tog ether, the results show that the rate constant for breaking a linear c ross-link formed by (DCT)(2)-cys is the same as that for dissociation of the monovalently bound (DCT)(2)-cys. Further, they show that openin g of a bond in a dimer ring for the F(ab')(2) fragment occurs with app roximately the same dissociation rate constant as opening a bond in a linear cross-link. This rate constant is about three times smaller tha n that observed with IgE, suggesting that steric strain is caused by a pposed Fc segments in cyclic IgE dimers. Such structural interference may affect the functional consequences of IgE-Fc epsilon RI aggregatio n on the cell surface.