MEMBRANE PERMEABILIZATION BY ALPHA-HELICAL PEPTIDES - A FLOW-CYTOMETRY STUDY

The permeabilization by alpha-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluo rescent and poorly membrane permeant molecule, becomes strongly fluore scent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by alpha-heli cal peptides such as melittin, succinylated melittin and anionic pepti des derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) caused a rapid (< 5 min) and dose-dependent (ED(50) = 0.5 mu M) perme abilization of HL60 cells at neutral pH, whereas the succinylated deri vative induced cell permeabilization only at pH below 4.5 with an ED(5 0) = 18 mu M. The permeabilization by the anionic ESCA peptide (GLFEAI AEFIEGGWEGLIEGCA) containing 5 glutamic residues occurred (ED(50) = 11 mu M) at pH ranging from 6.5 to 6.0; replacing the tryptophan residue in position 14 by a phenylalanine residue decreased by about 1 unit t he pH at which membrane permeabilization was effective. The membrane p ermeabilization activity of the E5CA peptide was reversibly abolished when the peptide was linked to a protein carrier. These results show t hat ct-helical peptide-induced membrane permeabilization can be easily monitored by using flow cytometry in the presence of a non permeant d ye. This method allows a rapid screening and an efficient mean of sele ction of peptides suitable to induce membrane permeabilization.