The significant diversity of lipid species and the manner which they are organized play a crucial role in the structure and function characteristics of the plasma membrane and other membrane-bound organelles. The asymmetric distribution of lipids in the inner and outer leaflets is a property of particular interest as it is believed to influence various cellular processes including vesicle budding and transport, signal transduction and the regulation of membrane-associated proteins. Although methods exist to create asymmetrical liposomes in vitro, their complexity pales in comparison to living cells, which have more lipid types, membrane proteins and ATP-dependant enzymes which actively maintain transbilayer asymmetry. In order to enhance this process of synthetically preparing biomimetic asymmetric membranes, optimizations in lipid characterization assays will be required. In this study two methods, 1H NMR and a spectrophotometric TNBS assay, are used to quantify the transbilayer asymmetry of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), respectively, at various mole percent concentrations in synthetic liposomes. By comparing the experimental signal of the external leaflet to the signal of the entire bilayer, the relative quantity of the different headgroup lipids can be quantitatively determined for each leaflet. The use of two tests enables complementary data to be collected to help ensure both are yielding accurate results. For example, in a sample containing a 1:1 POPC to POPE ratio, doped with 5% 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), the 1H NMR results indicated that 54.0% of the POPC was located in the outer leaflet and the TNBS assay results showed that 44.1% of the POPE was distributed in the outer leaflet, when the difference in leaflet size was corrected for. In addition to simply measuring the relative distribution of POPC and POPE, the effects of changing other experimental parameters, such as liposome size, temperature and the presence of cholesterol or other lipids, can be analyzed for their effects on the assays. Overall, the optimization of lipid asymmetry characterization assays is essential to further enhance the development of more realistic asymmetric model membranes.

Examination of the asymmetric transbilayer organization of phosphatidylcholine and phosphatidylethanolamine phospholipids in synthetic liposomes using 1H NMR and spectrophotometric analysis

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