The effects of hydrostatic pressure on the location of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), an environmentally sensitive fluorescent probe, in phosphatidylcholine lipid bilayers and in goldfish brain synaptic membranes have been studied by fluorescence spectroscopy over the pressure range of 0.001-2 kbar. The emission spectrum of PRODAN in all the membrane systems examined exhibits two local maxima: one centers at around 435 nm and the other at about 510 nm. The intensity ratio of these two peaks, F435/F510, increases as pressure increases; in the particular case of dimyristoyl-L-alpha-phosphatidylcholine multilamellar vesicles [DMPC(MLV)], a dramatic change in F435/F510 appears at the lipid phase transition pressure. As pressure varies, an isoemissive point is seen in both egg yolk phosphatidylcholines and goldfish brain synaptic membranes; however, no isoemissive point is observed in DMPC(MLV). The presence of an isoemissive point is attributed to a pressure-induced relocation of PRODAN from the "polar" disposition (the 510-nm peak) to the "less polar" disposition (the 435-nm peak). The absence of an isoemissive point in the case of DMPC(MLV) is probably due to the lack of void space in the lipid matrix, as a result of tight lipid packing. Apparently, the probe relocation takes place in unsaturated systems, and PRODAN favors a more hydrophobic environment under pressure. However, on the basis of the emission spectra, PRODAN seems to remain more or less at the interfacial region over the pressure range examined. In goldfish brain synaptic membranes, the PRODAN polarization increases with pressure, giving dT/dP values of 15-20 degrees C kbar-1 for both dispositions.(ABSTRACT TRUNCATED AT 250 WORDS)

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We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.

We have examined the effect of 1-palmitoyl-2-(10-pyrenyl)decanoyl-sn-glycerol-3-phosphatidylcholine (Pyr-PC) concentration on the ratio of excimer fluorescence to monomer fluorescence (E/M) in L-alpha-dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles at 30 degrees C, with special attention focussed on the smoothness of the curve. We observed a series of dips, in addition to kinks, in the plot of E/M versus the mole fraction of Pyr-PC (XPyrPC). The observation of dips is a new finding, perhaps unique for Pyr-PC in DMPC since only kinks were observed for Pyr-PC in L-alpha-dipalmitoylphosphatidylcholine (DPPC) and in egg yolk phosphatidylcholine (egg-PC) (Somerharju et al., 1985. Biochemistry. 24: 2773-2781). The dips/kinks observed here are distributed according to a well defined pattern reflecting a lateral order in the membrane, and distributed symmetrically with respect to 50 mol% Pyr-PC. Some of the dips appear at specific concentrations (YPyrPC) according to the hexagonal super-lattice model proposed by Virtanen et al. (1988. J. Mol. Electr. 4: 233-236). However, the observations of dips at XPyrPC > 66.7 mol% and the kink at 33.3 mol% cannot be interpreted by the model of Virtanen et al. (1988). These surprising results can be understood by virtue of an extended hexagonal super-lattice model, in which we have proposed that if the pyrene-containing acyl chains are regularly distributed as a hexagonal super-lattice in the DMPC matrix at a specific concentration YPyrPC, then the acyl chains of DMPC can form a regularly distributed hexagonal super-lattice in the membrane at a critical concentration (1-YPyrPC). The excellent agreement between the calculated and the observed dip/kink positions, except for the dip at 74 mol% and the kink at 40 mol%, provides most compelling evidence that lipids are regularly distributed into hexagonal super-lattices in Pyr-PC/DMPC mixtures at specific concentrations. The physical nature of the dips not only gives us a better understanding of lipid lateral organization in membranes but also will lead to new theoretical considerations and experimental designs for exploring the relationship between lipid regular distribution and membrane functions.

Steady-state fluorescence of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been employed to study the interacting effects between ethanol and pressure on the formation of the fully interdigitated dipalmitoylphosphatidylcholine (DPPC). At 1 atm and 20 degrees C, a dramatic change in the emission spectrum of Prodan fluorescence is observed at about 1.1-1.3 M ethanol. The emission maximum shifts to longer wavelengths, and the intensity ratio of Prodan fluorescence at 435 nm to that at 510 nm, F435/F510, decreases abruptly with increasing ethanol content. The spectral changes are correlated to the ethanol-induced phase transition of DPPC from the noninterdigitated gel state to the fully interdigitated gel state [Rowe, E.S. (1983) Biochemistry 22, 3299-3305; Simon, S.A., & McIntosh, T.J. (1984) Biochim. Biophys. Acta 773, 169-172]. The spectral changes are attributed to the probe relocation from a less polar environment to a more polar environment due to lipid interdigitation. This relocation is either due to the bulky terminal methyl group of the lipids or due to the partition of Prodan into the bulk solution or both. The present study demonstrates that Prodan is a useful probe in monitoring the formation of the ethanol-induced fully interdigitated DPPC gel phase. Pressure is found to produce spectral changes similar to those induced by ethanol when the ethanol content amounts to 0.8-1.1 M. At lower (e.g., less than 0.4 M) and higher ethanol (e.g., greater than 2.4 M) concentrations, pressure is unable to induce such spectral changes. The critical ethanol concentrations for the formation of the fully interdigitated DPPC gel phase (Cr) have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)