Effects of probe-amphiphile interaction on pyranine proton transfer reactions in lecithin vesicles

Photoacids and photobases are powerful tools for the discrimination, in real time, between the preferential pathways in H+/OH- transfer processes in biological and biomimetic systems which can determine the origins of crucial biochemical processes. In this study, the binding of 8-hydroxy-1,3,6-pyrenetrisulphonate sodium salt (pyranine) to phosphatidylcholine (lecithin) vesicles was investigated (by time-resolved and steady state fluorometry and laser-induced pH jump measurements) as a function of the probe and electrolyte concentration to determine how these parameters affect the properties of the photoacid. The ratio (R) between the fluorescence intensities at 510 nm (dissociated form) and 440 nm (undissociated form) can be related to the excited state degree of dissociation (D (%)). The binding of the probe to the internal leaflet of lecithin small unilamellar vesicles (SUVs) is larger than that to the external leaflet. The addition of salt up to 2 M does not prevent binding even at low probe concentrations. Time-resolved emission decay measurements also show the probe-lipid interaction. The lifetime of the undissociated form is equivalent to that in alcoholic solutions (approximately 3.8 ns). The pyranine ground state re-protonation rate constant (k(obs)) is dependent on the probe content per vesicle. With 550 pyranine molecules per vesicle, k(obs) is constant within a large range of salt concentration. In this situation, an alternative route is proposed (compared with direct PO-/H+ recombination), in which proton transfer between neighbouring species maintains an unchanging recombination rate. The data presented show that careful analysis of pyranine H+ transfer data is necessary for biomimetic and biological or reconstituted systems. (C) 1997 Elsevier Science S.A.