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Laurdan (6-dodecanoyl-2-dimethylamine-naphthalene) is a fluorescent membrane probe of recent characterization. It was shown that this probe discriminates between phase transitions, phase fluctuations and the coexistence of phase domains in phospholipid multilamellar aggregates. We measured the excitation and emission generalized polarization (GP(ex) and GP(em)) of Laurdan in aggregates of complex glycosphingolipids in their pure form and in mixtures with dipalmitoylphosphatidylcholine (DPPC). Our results show that Laurdan detects the broad main phase transition temperature of the neutral ceramide-tetrasaccharide Gg(4)Cer (asialo-G(M1)) and shows a value of GP(ex) in between that of DPPC and that of ganglioside G(M1). In contrast, Laurdan was unable to detect the thermotropic phase transition of G(M1). The probe also appears to be unable to detect phase coexistence in both types of pure glycolipid aggregates. Deconvolution of the excess heat capacity vs. temperature curves of pure Gg(4)Cer and DPPC/Gg(4)Cer mixtures indicates that the thermograms are composed by different transition components. For these cases, Laurdan detects only the high cooperativity component of the transition of the mixture. The peculiar behaviour of Laurdan in aggregates containing complex glycosphingolipids may result from the inherent topological features of the interface that are conferred by the bulky and highly hydrated polar head group of these lipids.

This report presents evidence that ibuprofen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a concentration as low as 10μM; b) in isolated unsealed human erythrocyte membranes (IUM) induced mild increase in the water content or in their molecular dynamics at the hydrophobic-hydrophilic interphase, while a corresponding ordering decrease at the deep phospholipids acyl chain level; c) at physiological temperature (37°C), 300μM ibuprofen induced a significant increase in the generalized polarization (GP) of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUV), an indication that ibuprofen molecules locate in the head polar group region of DMPC; d) X-ray diffraction studies showed that ibuprofen concentrations≥300μM induced increasing structural perturbation to DMPC bilayers; e) differential scanning calorimetry (DSC) data showed that ibuprofen was able to alter the cooperativity of DMPC phase transition in a concentration-dependent manner, to destabilize the gel phase and that ibuprofen did not significantly perturb the organization of the lipid hydrocarbon chains. Additionally, the effect on the viability of both human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cells was studied.

Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.