Pore kinetics reflected in the dequenching of a lipid vesicle entrapped fluorescent dye

Abstract

Pore formation in lipid vesicle membranes can be monitored by the fluorescence signal F(t) arising from the induced release of a self-quenching dye in the course of the elapsed efflux time t. We present a basic theoretical analysis of pertinent experimental data allowing the quantitative evaluation of information on the pore kinetics and mechanism. This implies an investigation of the 'dynamic' quenching factor Qt exhibited by that fraction of dye which is still being retained inside the liposomes at t. It is shown how Qt depends on the mode of release which could be 'all-or-none' or more gradual as expressed by a parameter rho < or = 1 (related to the pore lifetime), i.e., the average dye retention factor in a vesicle after a single pore opening. A fit to measured values of Qt at a sufficient extent of efflux may be applied in order to determine rho. Then the pore formation rate per liposome, va(t), can be derived from the registered F(t). We give a practical demonstration of the procedures with carboxyfluorescein-loaded phosphatidylcholine liposomes of two different sizes to which the wasp venom peptide mastoparan X had been added.