Studies of light emission, absorption and energy transfer in nerve membranes labelled with fluorescent probes

Physicochemical properties of fluorescent membrane probes, 2-p-Cl-anilinonaphthalene-6-sulfonate (p-Cl-ANS), 2-p-Br-anilinonaphthalene-6-sulfonate (p-Br-ANS), merocyanine-540, methyl violet, etc., were examined because of the possibility of demonstrating resonance energy-transfer between probes. The emission psectra of p-Cl-ANS and p-Br-ANS and the absorption (or fluorescence excitation) spectra of Eastman Kodak merocyanine-540 (M-540) and other probes were found to be very sensitive to changes in the solvent polarity. The spectra of these probes incorporated in the nerve membrane were determined and compared with the corresponding spectra in various organic solvents and macromolecules. This comparison suggests that the polarity of the binding sites for p-Cl-ANS (and p-Br-ANS) in the membrane is high and that of M-540 is very low. The spectrum of the portion of p-Cl-ANS fluorescence contributing to production of optical responses (i.e., transient changes during action potentials) was determined. Both absorption responses and fluorescence responses were detected from M-540 in the nerve membrane. It was possible to demonstrate resonance transfer of electronic energy from p-Cl-ANS to M-540 incorporated in lysolecithin micelles and in crab nerves. During action potentials, the intensity of M-540 fluorescence excited by energy transfer was found to undergo transient changes. Based on these and other experimental findings, properties of the binding sites for these probes in the nerve membranes are discussed.