CHARACTERIZATION OF PEPTIDE DIFFUSION INTO ELECTROPERMEABILIZED NEUTROPHILS

The superoxide (O-2(-))-generating NADPH oxidase of human neutrophils consists of membrane-bound and cytosolic proteins that assemble in the plasma membrane of activated cells. To date, most of our understandin g of the assembly of the NADPH oxidase has been obtained through the u se of a cell-free assay, and a number of peptides that mimic regions o f NADPH oxidase proteins have been shown to block oxidase assembly usi ng this assay. However, the cell-free assay provides an incomplete rep resentation of the assembly and regulation of the NADPH oxidase in viv o, and it has become necessary to develop methods for introducing biom olecules, such as peptides, into intact neutrophils where their effect s can be investigated. One such method is electropermeabilization. Alt hough this method has been used previously with human neutrophils, it has not been well characterized. We report here a detailed characteriz ation of the electropermeabilized neutrophil assay system, including o ptimal conditions for membrane electropermeabilization with maximal re tention of functional capacity, optimal conditions for analyzing the e ffects of experimental peptides, quantification of internalized peptid e concentration, and molecular size limits for diffusion of molecules into these cells. Our results demonstrate that optimal neutrophil perm eabilization (98-100%) can be achieved using significantly lower elect rical fields than previously reported, resulting in the retention of h igher levels of O-2(-)-generating activity. We also found that biomole cules as large as 2.3 kDa readily diffuse into permeabilized cells. An alysis of flavocytochrome b peptides that were shown previously to inh ibit NADPH oxidase activity in a cell-free assay demonstrated that the se peptides also blocked O-2(-) production in electropermeabilized hum an neutrophils; although at higher effective concentrations than in th e cell-free system. Thus, electropermeabilized neutrophils provide a m odel system for evaluating the effects of peptides and other pharmacol ogical agents in intact cells which closely mimic neutrophils in vivo.