LEAKAGE OF MEMBRANE VESICLE CONTENTS - DETERMINATION OF MECHANISM USING FLUORESCENCE REQUENCHING

Agents such as antimicrobial peptides and toxins can permeabilize memb rane vesicles to cause leakage of entrapped contents in either a grade d or an all-or-none fashion. Determination of which mode of leakage is induced is an important step in understanding the molecular mechanism of membrane permeabilization. Wimley et al. (1994, Protein Sci. 3:136 2-1378) have developed a fluorescence method for distinguishing the tw o modes that makes use of the dye/quencher pair 8-aminonapthalene-1,3, 6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) withou t the usual need for the physical separation of vesicles from released contents. Their ''requenching'' method establishes the mode of releas e through the fluorescence changes that occur when DPX is added extern ally to a solution of vesicles that have released some fraction of the ir contents. However, the requenching method as originally stated igno red the possibility of preferential release of dye or quencher. Here w e extend the theory of the method to take into account preferential re lease and the effects of graded leakage, The ratio of the rates of rel ease of the cationic quencher DPX and anionic dye 8-aminonapthalene-1, 3,6 trisulfonic acid can be estimated by means of the theory. For grad ed leakage, we show that the release of the markers does not coincide with the fluorescence changes observed in the standard leakage assay. This is true for self-quenching dyes as well and means that 1) the amo unt of released material will be overestimated and 2) the kinetics wil l be nonexponential and have artificially high apparent rates. We show how the extended requenching analysis allows the results of leakage e xperiments to be corrected for artifacts that result from graded and p referential leakage. Experimental evidence is presented for the existe nce of peptide-induced preferential graded leakage of DPX from both ne utral and anionic vesicles.