PHYSICOCHEMICAL DETERMINANTS OF THE SENSITIZING EFFECTIVENESS FOR PHOTOOXIDATION OF NERVE MEMBRANES BY FLUORESCEIN DERIVATIVES

Abstract— Fluorescein and 8 of its halogenated derivatives were employed as sensitizers for the photochemical block of sodium channels on giant nerve axons from lobsters. Axons were voltage clamped using a double sucrose gap technique and illuminated by filtered light from a Xe arc following exposure to sensitizer at concentrations of 5 µ M or less dissolved in the external bath. An assay for the magnitude of sensitizing effectiveness was developed consisting of the rate constant for block of channels during illumination scaled to the rate of photon capture in a thin layer of bathing solution. The kinetics for development of effectiveness during pre‐illumination periods of up to 16‐min duration varied widely for different derivatives despite similarities in molecular structure. At a standard 8‐min pre‐illumination exposure, the relative effectiveness extended over a 30,000‐fold range. A model system consisting of octanol and the artificial sea‐water (ASW) bathing solution was compared with the axon membrane and its environment. Octanol/ASW partition coefficients were measured for each sensitizer as well as the relative absorption in the 2 solvents at equal concentrations. Predicted values of relative sensitizing effectiveness based on the product of partition coefficient, relative absorption in octanol and mol wt of the substituents on the fluorescein skeleton were found to correlate highly with measured values. The diffusion kinetics prior to illumination correlated inversely with partition coefficients, suggesting that the more permeable sensitizers may penetrate into diffusion sinks, thus slowing the rise in concentration at sites for modification. It is concluded that sensitizers must penetrate into the membrane in order to sensitize and that the photochemistry appropriate for a low polarity environment controls the degree of membrane modification following photon capture.