RELATIVE BINDING-PROPERTIES OF FLUORESCEIN AND 9-HYDROXYPHENYFLUORON (HPF) WITH MURINE MONOCLONAL ANTIFLUORESCEIN ANTIBODIES

Contribution of the fluorescein (F1) carboxyl group to hapten binding by idiotypically related murine monoclonal anti-F1 antibodies 4-4-20, 9-40 and 12-40 was studied by comparing relative liganded active site properties with bound Fl or 9-hydroxyphenylfluoron (HPF). Kinetic studies revealed similar association rate constants between Fl and HPF to 4-4-20 (almost-equal-to 1.1 . 10(7) M-1 s-1); however, the 4-4-20 dissociation rate for Fl was almost-equal-to 200 times slower, relative to HPF, which resulted in relative intrinsic affinity values of 1.2 . 10(10) and 6.5 . 10(7) M-1, respectively. Mabs 9-40 and 12-40 also displayed a reduced affinity for HPF and affinity constants of 5.5 . 10(5) M-1 and 6.7 . 10(5) M-1 were obtained from a competitive ELISA. Additionally, previous studies revealed that upon binding Fl, Mabs 4-4-20 (92.1%), 9-40 (44.7%) and 12-40 (73.4%) quenched Fl fluorescence. Similar analyses with HPF resulted in 64.4% and 2.0% fluorescence quenching by 4-4-20 and 12-40, respectively; however, 9-40 increased HPF fluorescence by almost-equal-to 24%. Steady-state fluorescence polarization experiments revealed that in solution, Fl (P = 0.019) and HPF (P = 0.048) were polarized to different degrees. When bound, however, Fl and HPF expressed similar polarization values (P almost-equal-to 0.455), except 9-40 bound HPF which was significantly depolarized (P = 0.428). Fluorescence lifetime experiments revealed Fl to possess two discrete lifetimes: a 3.96 ns component (free Fl) and either a 0.52 ns (4-4-20), 2.23 ns (9-40) or 0.96 ns (12-40) short component that corresponded to bound Fl. HPF, however, when bound by 4-4-20 or 9-40, was best fit by three discrete exponentials: a relatively long 4.0 ns component, a 1.11 ns lifetime (free HPF) and either a 0.52 ns (4-4-20) or 2.23 ns (9-40) component. Finally, HPF bound by Mab 12-40 exhibited a single lorenzian distributed lifetime of 1.36 ns (+/- 0.43 ns). Results are discussed in terms of Mab active site structure and conformational state dynamics.