DISTANCE BETWEEN CYS-2O1 IN ERYTHROCYTE BAND-3 AND THE BILAYER MEASURED BY SINGLE-PHOTON RADIOLUMINESCENCE

Single-photon radioluminescence (SPR), the excitation of fluorophores by short-range P-decay electrons; was developed for the measurement of submicroscopic distances, The cytoplasmic domain of band 3 (cdb3) is the primary, multisite anchorage for the erythrocyte skeleton, To begi n to define the membrane arrangement of the highly asymmetrical cdb3 s tructure, the distance from the bilayer of Cys-201 next to the ''hinge '' of cdb3 was measured by both SPR and resonance energy transfer (RET ), cdb3 was labeled at Cys-201 with fluorescein maleimide. For SPR mea surements, the bilayer was labeled with [H-3]oleic acid, The corrected cdb3-specific SPR signal was 98 +/- 2 cps mu Ci(-1) [mu mol band 3](- 1). From this and the signal from a parallel sample in which (H2O)-H-3 was substituted for [H-3]oleic acid to create uniform geometry betwee n H-3 and the fluorophores, a Cys-201-to-bilayer separation of 39 +/- 7 Angstrom was calculated. Confirmatory distances of 40 and 43 Angstro m were obtained by RET between fluorescein on Cys-201 and eosin and rh odamine B lipid probes, respectively. This distance indicates that Cys -201 lies near band 3's vertical axis of symmetry and that the subdoma in of cdb3 between the hinge and the membrane is not significantly ext ended. In addition, these results validate SPR as a measure of molecul ar distances in biological systems.