Single photon radioluminescence. II. Signal detection and biological applications

Abstract

A quantitative theory for excitation of fluorescent molecules by beta decay electrons is reported in the accompanying manuscript; experimental detection methods and biological applications are reported here. The single photon signals produced by an excited fluorophore (single photon radioluminescence, SPR) provide quantitative information about the distance between radioisotope and fluorophore. Instrumentation was constructed for SPR signal detection. Photons produced in a 0.5-ml sample volume were detected by a cooled photomultiplier and photon counting electronics. To minimize electronic noise and drift for detection of very small SPR signals, a mechanical light chopper was used for gated-signal detection, and a pulse height analyzer for noise rejection. SPR signals of approximately 1 cps were reproducibly measurable. The influence of inner filter effect, sample turbidity, and fluorophore environment (lipid, protein, and carbohydrate) on SPR signals were evaluated experimentally. SPR was then applied to measure lipid exchange kinetics, ligand binding, and membrane transport, and to determine an intermolecular distance in an intact membrane. (a. Lipid exchange kinetics.) Transfer of 12-anthroyloxystearic acid (12-AS) from sonicated lipid vesicles and micelles to vesicles containing 3H-cholesterol was measured from the time course of increasing SPR signal. At 22 degrees C, the half-times for 12-AS transfer from vesicles and micelles were 3.3 and 1.1 min, respectively. (b. Ligand binding.) Binding of 3H-oleic acid to albumin in solution, and 3H-2,2'-dihydro-4,4'-diisothiocyanodisulfonic stilbene (3H-H2DIDS) to band 3 on the erythrocyte membranes were detected by the radioluminescence of the intrinsic tryptophans. The SPR signal from 5 microCi 3H-oleic acid bound to 0.3 mM albumin decreased from 13 +/- 2 cps to 3 +/- 2 cps upon addition of nonradioactive oleic acid, giving 2.7 high affinity oleic acid binding sites per albumin. The SPR signal from 1 microCi 3H-H2DIDS bound selectively to erythrocyte band 3 in erythrocyte ghosts (1.5 mg protein/ml) was 2.2 +/- 0.8 cps. (c. Membrane transport). Dilution of J774 macrophages loaded with 3H-3-O-methylglucose and BCECF gave a decreasing SPR signal with a half-time of 81 s due to methylglucose efflux; the SPR measurement of the efflux rate was in agreement with a conventional tracer efflux rate determination by filtration. 20 microM cytochalasin B inhibited efflux by 97%. (d. Distance determination.) The SPR signal from erythrocyte membranes labeled with 27 microCi 3H-oleic acid and 10 microM of fluorescein-labeled wheat germ agglutinin was 5.7 +/- 0.5 cps, giving an average glycocalyx-to-bilayer distance of 5 nm. The results establish methods for experimental detection of SPR signals and demonstrate the applications of radioluminescence to the measurement of lipid exchange kinetics, ligand binding, membrane transport, and submicroscopic distances in intact membranes in real time.