New fluorescent cholesterol analogs as membrane probes

Abstract

New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.

Behavior of GM3 ganglioside in lipid monolayers mimicking rafts or fluid phase in membranes

“…5A shows the (Π-A) isotherms of POPC-GM 3 mixtures at different molar ratios. Pure POPC monolayer is in an expanded state, in agreement with previous reports (Lai et al, 1994;Grechishnikova et al, 1999), and fluorescence images of POPC monolayers are completely uniform within the surface pressure range 0-35 mN/m (data not shown).…”

Oxidation of Langmuir-Blodgett films of monounsaturated lipids studied by atomic force microscopy

“…These isotherms are on the whole similar, showing in particular that both lipids are in a liquid-expanded phase during all the compression under our experimental conditions. These results are on the whole in agreement with previously published isotherms, obtained under similar experimental conditions [36,37], in particular at temperatures higher than the transition temperatures of these lipids, around -3°C and -9°C for POPC and OPPC, respectively [38][39][40]. Figure 3 shows AFM phase images of POPC and OPPC LB films just after their transfer at 30 m.Nm -1 on freshly cleaved mica (J1).…”

Influence of oxidized lipids on palmitoyl-oleoyl-phosphatidylcholine organization, contribution of Langmuir monolayers and Langmuir–Blodgett films

“…2A shows the p A isotherms of PoxnoPC, POPC and their mixtures containing from 4 to 20 mol% of PoxnoPC. Isotherms of PoxnoPC and POPC are in agreement with those previously described (Sabatini et al, 2006;Lai et al, 1994;Grechishnikova et al, 1999), and suggest that these lipids are in liquid expanded phase, as their mixtures. The collapse surface pressure for PoxnoPC is rather low, around 31 mN m 1 , but the presence of POPC into the monolayers increases their stability at high pressure.…”