Dithionite Quenching Rate Measurement of the Inside−Outside Membrane Bilayer Distribution of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Phospholipids†

Measurement of the extent of dithionite quenching of the fluorescence of 7-nitrobenz-2-oxa-1,3-diazol-4-yl- (NBD-) labeled lipids inserted into cellular and organellar membranes has been used to quantify their topological distribution and translocation. This assay provides a straightforward method for determining the fraction of NBD-lipid exposed to the outer leaflet of membranes that are impermeant to dithionite. However, it appears that many, if not all, cellular membranes are relatively permeable to dithionite. The present work describes a method in which the initial rate of dithionite quenching, rather than the extent of quenching, was used to determine the fraction of different NBD-labeled phospholipids exposed to the outer leaflet. This method permits the estimation of the translocation process even in experimental conditions where the membrane is semipermeable to dithionite. This technique was used to measure the translocation of several NBD-labeled phospholipids across two biological membranes:  b...