Influence of Structural Heterogeneity on the Fluorimetric Response Characteristics of Lipid Membranes Containing Nitrobenzoxadiazolyldipalmitoylphosphatidylethanolamine

Structural alterations in lipid membranes that are induced by changes in pH or by selective enzyme−substrate or antibody−antigen interactions can provide changes in fluorescence intensity from membranes containing nitrobenzoxadiazolyldipalmitoylphosphatidylethanolamine (NBD−PE). For example, fluorimetric responses of vesicles consisting of mixtures of phosphatidic acid (PA) and phosphatidylcholine (PC) prepared either from egg yolk or as synthetic, dipalmitoyl-substituted moieties and containing 2.3 mol % NBD−PE consist of either an increase or decrease in total fluorescence yield upon alteration of supporting electrolyte pH. This response was due to changes in NBD−PE self-quenching and the local environment of the fluorophore. The present work attempts to identify the factors that determine the fluorescence sensitivity of these lipid membranes. The impact of phosphatidic acid loading in vesicular bilayer lipid membranes and microscopic structural heterogeneity in monolayers at the air−water interface were explored. The results indicated that monolayers that exhibited laterally-separated phase domains on the microscopic scale provided enhanced sensitivity to perturbations of bulk pH and membrane surface charge redistribution through the alteration of the degree of headgroup ionization. Variations in the surface potential upon compression of either egg PA/egg PC or DPPA/DPPC monolayers in the liquid-expanded state were observed in order to facilitate the qualitative correlation of trends in the fluorimetric response of structurally homogeneous and structurally heterogeneous vesicular membranes with the existence of laterally-separated phase domains. The results indicated a strong correlation between sensitivity and structurally heterogeneity. The results also suggest the existence of ordered sub-microscopic domains in otherwise structurally homogeneous egg PA/egg PC membranes.