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The Lipid Dependence of Melittin Action Investigated by Dual-Color Fluorescence Burst Analysis

Geert van den Bogaart ^*, Jacek T. Mika ^*, Victor Krasnikov  and Bert Poolman ^*

^* Biochemistry Department, and Ultrafast Laser and Spectroscopy Laboratory, Groningen Biomolecular Science and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands

Correspondence: Address reprint requests to Bert Poolman, Tel.: 31-50-363-4190; Fax: 31-50-363-4165; E-mail: b.poolman{at}rug.nl.

Dual-color fluorescence-burst analysis was used to study melittin-induced leakage of macromolecules from liposomes of various lipid compositions. To perform dual-color fluorescence-burst analysis, fluorescently labeled size-marker molecules were encapsulated into liposomes, labeled with a second lipid-attached fluorophore. By correlating the fluorescence bursts, resulting from the liposomes diffusing through the detection volume of a dual-color confocal microscope, the distribution of size-marker molecules over the liposomes was determined. It was found that melittin causes leakage via two different mechanisms: 1), For liposomes composed of neutral bilayer-forming lipids, low melittin concentrations induced pore formation with the pore size depending on the melittin concentration. 2), For liposomes containing anionic and/or nonbilayer forming lipids, melittin induced fusion or aggregation of liposomes accompanied by a-specific leakage. Experiments with liposomes prepared from Escherichia coli lipid extracts and intact cells of Lactococcus lactis indicate that both mechanisms are physiologically relevant.