Assay for the transbilayer distribution of glycolipids. Selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine- egg phosphatidylethanolamine;-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A(2) confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing