The use of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipids in determining transmembrane lipid distribution.
Transbilayer lipid distribution of small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) was measured using 31P-nuclear magnetic resonance (NMR) spectroscopy, chemical modification with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dithionite reduction of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipid (NBD-lipid). The dithionite assay was the most reproducible of the three assays, with 1.2% error for SUVs and 3.9% error for LUVs. The dithionite assay also agreed best with theoretical inner:outer leaflet ratios, based on vesicle diameters determined by electron microscopy (Thomas et al. (1989) Biochem. Biophys. Acta 978, 85-90). Dithionite assay measurements were within 2.7% of theoretical ratios for SUVs and 2.3% for LUVs, while the NMR assay for SUVs was 14% lower than theoretical ratios and 23% lower for LUVs. The accuracy of NBD-lipids as markers for total transbilayer lipid was investigated. NBD-labeled phosphatidylserine, phosphatidylcholine and phosphatidylglycerol were accurate markers for total transbilayer lipid distribution, as their distributions were in close agreement with theoretical ratios. However, NBD-labeled phosphatidylethanolamine displayed a slight preference for the inner leaflet at low mole fractions of phosphatidylethanolamine, while native phosphatidylethanolamine showed a preference for the outer leaflet at the same concentration. NBD-labeled phosphatidic acid also showed a slight preference for the inner leaflet. We conclude that although dithionite-based assessment of NBD-labeled lipids across membrane bilayers can be a powerful analytical tool, caution must be used in the interpretation of results.
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Ohio 45267-0524.
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D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D004227:Dithionite / Q000032:analysis; D005456:Fluorescent Dyes; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000032:analysis*; D008081:Liposomes / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008854:Microscopy, Electron; D010714:Phosphatidylethanolamines; D010758:Phosphorus; D015203:Reproducibility of Results; D014302:Trinitrobenzenesulfonic Acid
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D004227:Dithionite; D010758:Phosphorus; D014302:Trinitrobenzenesulfonic Acid; D009327:4-Chloro-7-nitrobenzofurazan