Phosphatidic acid affects structural organization of phosphatidylcholine liposomes. A study of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) fluorescence decay using distributional analysis

The fluorescence decay of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to study micro-heterogeneity of 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) liposomes and to characterize the effect of phosphatidic acid on the correlation between fluorescence microheterogeneity and membrane permeability. The fluorescence decay, measured using multifrequency phase fluorometry, has been analyzed either by using a model of discrete exponential components or a model of continuous distribution of lifetime values. Both analyses have shown that TMA-DPH decay is characterized by two components: a long one of about 9 ns and a short one of about 5 ns. In the gel phase, at variance with previous DPH studies, the short component was associated with a large fractional intensity. The distributional analysis showed changes of lifetime values and width in correspondence to the calorimetric transitions. The presence of egg phosphatidic acid increased both long lifetime values and distributional width. The use of TMA-DPH as a probe to evaluate membrane heterogeneity using the distributional width is discussed. The effect of phosphatidic acid on the membrane surface and in the hydrophobic core has been related to its structural properties and to its role in water penetration.     

Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers.

The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37 degrees C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased membrane heterogeneity in stimulated human granulocytes

TMA-DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non-stimulated granulocytes TMA-DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes and redistribution of membrane components.     

Multiple conformational states in myoglobin revealed by frequency domain fluorometry

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membranes modification of differentiating proerythroblasts. Variation of 1,6-diphenyl-1,3,5-hexatriene lifetime distributions by multifrequency phase and modulation fluorimetry

The fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) in K562 cell membranes has been studied using multifrequency phase and modulation fluorimetry. The DPH decay data collected at various modulation frequencies were analysed by assuming either a model of discrete exponential components or a model of continuous lifetime distribution. The fits showed smaller values of the reduced chi square using the model of continuous lifetime distribution. The K562 cell membranes dynamics were investigated during the cell differentiation along the erythroid pathway. By using the continuous lifetime distribution method for the analysis of the DPH decay, marked variations were observed during the four initial days of the erythroid differentiation. Namely, the width of the DPH lifetime distribution increased by a factor of about two, while the center value of the distribution remained constant. By using the discrete exponential components model for the analysis of the DPH decay no variations were observed during the K562 differentiation.     

A photophysical model for diphenylhexatriene fluorescence decay in solvents and in phospholipid vesicles.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in pure solvents and in phospholipid vesicles has been measured using frequency domain fluorometry. Data analysis uses a model with two energetically close excited states. The model explains the high quantum yield and the double exponential decay of DPH observed in some pure solvents and in phospholipid vesicles. This model assumes that after excitation to a first excited state, there is a rapid interconversion to a lower excited state and that most of the emission occurs from this state. The interconversion rates between the two excited states determine the average lifetime. For DPH in solvents, we find that the interconversion rates are solvent and temperature dependent. For DPH in phospholipid vesicles, we find that the back reaction rate from excited state 2 to excited state 1 (R12) is what determines the fluorescence properties. The phospholipid phase transition affects only this back reaction rate. The model was analyzed globally for a range of solvents, temperatures and vesicle composition. Of the six parameters of the model, only two, the interconversion rates between the two excited states, varied in all different samples examined. For DPH in phospholipid vesicles, there is an additional feature of the model, which is related to the apparent distribution of the rate R12. Significantly better fits were obtained using a continuous lorentzian distribution of interconversion rates. The resulting lifetime distribution was asymmetric and showed a definite narrowing above the phase transition.     

Abscisic acid-induced microheterogeneity in phospholipid vesicle: a fluorescence study

Changes in the thermal behavior of DMPC (dimyristoyl-L-phosphatidylcholine) and an equimolar mixture of DMPC and DMPE (dimyristoyl-L-phosphatidylethanolamine) induced by the plant hormone abscisic acid (ABA) have been investigated using fluorescent probes. The fluorescence decay of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in these vesicles has been measured using frequency-domain fluorometry, and has been analyzed using both models of discrete exponential components and continuous lifetime distributions. In the DMPC vesicles, using the distributional approach, higher center and width values were observed in the presence of abscisic acid (ABA), indicating a decrease in the dielectric constant of the lipid phase that we attribute to a decrease in the water concentration within the bilayer. Moreover, the presence of ABA in the liposomes increased the phospholipid phase transition temperature. The addition of ABA to the DMPC/DMPE mixture strongly increased the microheterogeneity of the system as reported by the FWHM (full-width at half-maximum) of the distributional approach.     

Fluorescence lifetime distributions of diphenylhexatriene-labeled phosphatidylcholine as a tool for the study of phospholipid-cholesterol interactions.

Fluorescence lifetimes of 1-palmitoyl-2-diphenylhexatrienylpro-pionyl-phosphatidylc hol ine in vesicles of palmitoyloleoyl phosphatidylcholine (POPC) (1:300, mol/mol) in the liquid crystalline state were determined by multifrequency phase fluorometry. On the basis of statistic criteria (chi 2red) the measured phase angles and demodulation factors were equally well fitted to unimodal Lorentzian, Gaussian, or uniform lifetime distributions. No improvement in chi 2red could be observed if the experimental data were fitted to bimodal Lorentzian distributions or a double exponential decay. The unimodal Lorentzian lifetime distribution was characterized by a lifetime center of 6.87 ns and a full width at half maximum of 0.57 ns. Increasing amounts of cholesterol in the phospholipid vesicles (0-50 mol% relative to POPC) led to a slight increase of the lifetime center (7.58 ns at 50 mol% sterol) and reduced significantly the distributional width (0.14 ns at 50 mol% sterol). Lifetime distributions of POPC-cholesterol mixtures containing greater than 20 mol% sterol were within the resolution limit and could not be distinguished from monoexponential decays on the basis of chi 2red. Cholesterol stabilizes and rigidifies phospholipid bilayers in the fluid state. Considering its effect on lifetime distributions of fluorescent phospholipids it may also act as a membrane homogenizer.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

Advantages and limitations of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3- sn-phosphatidylcholine as a fluorescent membrane probe

We have investigated the behavior of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn -phosphatidylcholine (DPHpPC) in synthetic, multilamellar phosphatidylcholine vesicles. This fluorescent phospholipid has photophysical properties similar to its parent fluorophore, diphenylhexatriene (DPH). DPHpPC preferentially partitioned into fluid phase lipid (Kf/s = 3.3) and reported a lower phase transition temperature as detected by fluorescence anisotropy than that observed by differential scanning calorimetry. Calorimetric measurements of the bilayer phase transition in samples having different phospholipid to probe ratios demonstrated very slight changes in membrane phase transition temperature (0.1-0.2 degree C) and showed no measurable change in transition width. Nonetheless, measurements of probe fluorescence properties suggested that DPHpPC disrupts its local environment in the membrane and may even induce perturbed probe-rich local domains below the phospholipid phase transition. Temperature profiles of steady-state fluorescence anisotropy, limiting anisotropy, differential tangent, and rotational rate were similar to those of DPH below the main lipid phase transition but indicated more restricted rotational motion above the lipid phase transition temperature. As for DPH, the fluorescence decay of DPHpPC could be described by either a single or double exponential both above and below the DPPC phase transition. The choice seemed dependent on the treatment of the sample. The intensity-weighted average lifetime of DPHpPC was roughly 1.5 ns shorter than that of DPH. In summary, the measured properties of DPHpPC and its lipid-like structure make it a powerful probe of membrane structure and dynamics.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Membrane structural domains. Resolution limits using diphenylhexatriene fluorescence decay.

Measurement of multiple fluorescence decay times of 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes can in principle be used to investigate structural domains of lipid bilayers. To assess the feasibility of this approach using phase and modulation techniques, we reduced experimental errors specifically associated with performing these measurements on membrane suspensions (probe self-quenching, background fluorescence, turbidity-induced artifacts) and determined empirically the level of precision thereby obtainable. Next we used these precision limits in theoretical calculations to conclude that the ratio of two coexisting decay times must exceed 1.3 if they are to be resolved with reliable accuracy. To demonstrate that such resolutions could be accomplished experimentally in membrane suspensions, three approaches were taken. First, the fluorescence decay of aqueous quinine sulfate quenched by chloride ion was resolved from that of membrane-associated DPH as long as the lifetime ratios of these two fluorophores exceeded the predicted value. Second, populations of DPH-containing lipid vesicles with single (or nearly single) decay times were mixed together, and when there were only two major lifetime components that differed by more than 30%, the resulting heterogeneous fluorescence could be resolved into the two expected lifetime components. Finally, DPH fluorescence decay measurements were correlated with phase behavior in well-characterized lipid systems, revealing a short lifetime component of DPH fluorescence associated with gel-phase lipid vesicles. From these studies, we conclude that only in special cases can co-existing gel and fluid phases be resolved by means of DPH lifetime heterogeneity, within the limits of precision defined herein.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

Interaction of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine-d54 mixtures with glycophorin. A fourier transform infrared investigation

Glycophorin from the human erythrocyte membrane has been isolated in pure form and reconstituted into large unilamellar vesicles comprised of binary mixtures of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) and chain perdeuterated 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC-d54). The effect of temperature and protein on lipid structure and mixing was monitored by using Fourier transform infrared spectroscopy; deuteration of one of the components of the mixture permits observation of the protein interaction with each lipid species. The melting curves were analyzed by assuming that each lipid chain can exist in one of two physical states (i.e., gel or liquid crystalline), characterized by a temperature-dependent Lorentzian distribution for the line shape of the C-H or C-D stretching vibrations. The fraction of each lipid component melted at temperatures within the two-phase region of the phase diagram was calculated and approximate phase diagrams were constructed. Addition of protein lowers the liquidus line of the phase diagram while leaving the solidus line essentially unchanged. No lipid phase separation is observed. The effect of protein is more pronounced on the DPPC component than on the DMPC-d54. The former is significantly more disordered and/or fluidized at all lipid mole fractions in the ternary system than in the binary phospholipid mixture.     

Lipid domain structure correlated with membrane protein function in placental microvillus vesicles

Membrane fluidity properties of placental microvillus membrane vesicles (MVV) were determined from fluorescence anisotropy (r), dynamic depolarization, and lifetime heterogeneity studies of diphenylhexatriene (DPH), trimethylamino-DPH (TMA-DPH), and cis- and trans-parinaric acids (c-PnA and t-PnA). Plots of r against temperature for DPH and TMA-DPH in MVV had slope discontinuities at 26 degrees C (Tc, transition temperature); however, analysis of r in terms of probe rotational rate (R), limiting anisotropy (r infinity), and lifetime (tau) revealed that DPH reported a phase transition because of changes in r infinity, whereas the phase transition observed by TMA-DPH occurred primarily because of changes in R. Heterogeneity analysis using phase and modulation lifetimes at three frequencies showed that DPH and TMA-DPH lifetimes were homogeneous in MVV. Both long (greater than 25 ns) and short (less than 6 ns) lifetime components were detected for c-PnA and t-PnA in MVV, corresponding to the probes in solid and fluid lipid phases. The fractional amplitude of the long lifetimes (solid phase) decreased from 0.86 to 0.12 with increasing temperature (5-55 degrees C) as the membrane passed through the phase transition, with 50% of the change occurring at 27 degrees C (c-PnA) or 33 degrees C (t-PnA). The activation energies for alkaline phosphatase, aminopeptidase M, and sodium-proton antiporter activities all showed discontinuities in the temperature range 27-31 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

Short-lived fluorescence component of DPH reports on lipid--water interface of biological membranes

Fluorescence measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) in large unilamellar phospholipid vesicles were performed to characterize the influence of the membrane physical properties on the short-lived lifetime component of the fluorescence decay. We have found that the short-lived component of DPH significantly shortens when the membrane undergoes a temperature-induced phase transition as it is known for the long-lived component of DPH. We induced membrane phase transitions also by alcohols, which are reported to be distributed different way in the membrane–ethanol close to the membrane-water interface and benzyl alcohol in the membrane core. A different effect of the respective alcohol on the short and long decay component was observed. Both the time-resolved fluorescence spectra of DPH taken during lipid vesicle staining and the lifetime dependences caused by changes of temperature and/or induced by the alcohols show that the short-lived fluorescence originates from the population of dye molecules distributed at the membrane–water interface.     

Time-dependent changes in the size distribution of distearoylphosphatidylcholine vesicles.

The results of transmission electron microscopic and ultracentrifugal studies of the size distributions of sonicated distearoylphosphatidylcholine vesicles are reported. Small vesicles (d approximately 300 A) were prepared by sonication of pure 1,2-distearoyl-3-sn-phosphatidylcholine in water and incubated at 4, 21, 40, 53, and 65 degrees C. The vesicle size distributions changed as a function of time at all temperatures below the phase-transition temperature but remained constant at the transition temperature and above. The sizes of structures to which the small vesicles are converted are the same at all temperatures, although the rates of conversion differ. The primary structures formed are identified as larger vesicles. The rate of loss of small vesicles is found to increase with decreasing temperature. At 4 and 21 degrees C small vesicles are converted to amorphous material, possibly irregular fragments of neat phase, in addition to being converted to larger vesicles. Trace amounts of an impurity commonly produced in the synthesis of 1,2-distearoyl-3-sn-phosphatidylcholine, 1,3-distearoyl-2-sn-phosphatidylcholine, are found to dramatically reduce the rate of loss of small vesicles at 21 degrees C.     

Properties influencing fluorophore lifetime distributions in lipid bilayers

The fluorescence lifetime of the membrane fluorophore 1,6-diphenyl-1,3,5-hexatriene has been analyzed according to the distributional approach in a number of lipid bilayer systems. The systems included vesicles of 16:0/18:1-phosphatidylcholine (POPC), egg phosphatidylcholine (EYPC), microsomal phospholipids, and also intact microsomal membranes. With increasing complexity of composition, an increasingly broader width was found in the major component of a bimodal Lorentzian fluorescence lifetime distribution. In order to explain these findings, we propose a model based on environmental heterogeneity and environmental sampling, where the environment is defined as the lipid molecules immediately surrounding the fluorophore. Environmental heterogeneity is thought of as arising from organizational, compositional, and solvent factors. Environmental sampling pertains to the ability of a fluorophore to detect environments in a system and is a function of the fluorophore lifetime and the lipid dynamics. If the fluorescence lifetime is sufficiently short, the fluorophore will only sample a particular environment, and great compositional complexity will mean that each fluorophore in an ensemble will decay to the ground state with a different time. This appears to explain why in our results with DPH a narrow width is obtained for POPC, where vesicles are composed of a single phospholipid molecular species, compared to EYPC and microsomal phospholipid vesicles having complex molecular species composition. This model should serve as a basis for understanding the interrelationships of environmental complexity and lipid dynamics in membranes.     

Molecular organization and dynamics of the outer membrane of Salmonella thyphimurium mutant strains detected by frequency domain fluorometry

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the outer membrane bilayer of two mutant strains of Salmonella thyphimurium, i.e., SH 5014 and SH 6261, at different temperatures was analyzed in terms of continuous Lorentzian lifetime distributions. The results were compared with those obtained for the free fluorophore in an isotropic nonviscous solvent. The incorporation of DPH in the outer membrane fragments resulted in a broadening of the lifetime distribution which was attributed to the microenvironmental heterogeneity of the membrane bilayer for the extrinsic fluorophore. The differences observed between the two types of membrane bilayers were interpreted in terms of a different molecular organization and, to a lesser extent, in terms of a different fluidity. The comparison between the DPH lifetime distributions obtained using two different excitation wavelengths, i.e., 280 and 350 nm, suggested that the structural organization of the membrane domains, which are richest in proteins, is almost identical in the two examined mutant strains. This observation indicates that the different susceptibility of the two mutant strains toward phagocytosis and complement-mediated lytic action may depend on the molecular organization and dynamics of the lipid regions far from those containing proteins.