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.     

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)     

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.     

Analysis of cell membrane micro-heterogeneity using the fluorescence lifetime of DPH-type fluorophores.

Heterogeneity in the lipid organization in lipid bilayers and cell membranes was probed by using the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 position of phosphatidylcholine (DPH-PC). In the presence of protein, it is proposed that the bulk lipids and boundary lipids can potentially provide distinct enough fluorophore environments for two different lifetime centers to be recovered from the analysis of the fluorescence decay. To test this model experiments were performed with cytochrome b5 in 1-palmitoyl-2-oleoylphosphatidylcholine bilayers. The number of boundary lipids of cytochrome b5 is known from the literature or can be calculated from known dimensions, so that for a known protein:lipid ratio the fraction of lipids in the bulk and boundary lipid regions is known. These values were found to closely correspond to the fractions associated with the lifetime centers recovered from an analysis of the fluorescence decay assuming two major fluorophore populations. This indicated that the DPH distributed in a similar manner to the lipids and that its boundary lipid residency time was greater than the excited state lifetime, showing the validity of the approach. An important requirement was that the protein should influence the fluorophore decay sufficiently enough to enable separate lifetime centers for the bulk and boundary lipid fluorophores to be recovered by the analysis. Attempts were made to analyze the fluorescence decay of DPH in liver plasma membranes and microsomes as arising from two distinct fluorophore populations, however, the basic condition was not satisfied. By contrast, using DPH-PC it was possible to extract two separate lifetime centers. The limitations and potential of this approach are critically assessed and it is concluded that in certain circumstances information pertaining to the protein-lipid interfacial region of membranes can be extracted from fluorescence decay heterogeneity properties.     

Fluorescence energy transfer from diphenylhexatriene to bacteriorhodopsin in lipid vesicles

Fluorescence energy transfer between the donor diphenylhexatriene (DPH) and the acceptor retinal and fluorescence depolarization of DPH are used to test current theories for fluorescence energy transfer in two-dimensional systems and to obtain information on the effect of the intrinsic membrane protein, bacteriorhodopsin, on the order and dynamics of the lipid phase. Increasing the surface concentration of acceptors by raising the protein to lipid ratio leads to a decrease in the mean fluorescence lifetime by up to a factor of four. When the acceptor concentration is reduced at a fixed protein to lipid ratio by photochemical destruction of retinal, the lifetime increases and reaches approximately the value observed in protein-free vesicles when the bleaching is complete. The shape of the decay curve and the dependency of the mean lifetime on the surface concentration of acceptors are in agreement with theoretical predictions for a two-dimensional random distribution of donors and acceptors. From this analysis a distance of closest approach between donors and acceptors of approximately 18 A is obtained, which is close to the effective radius of bacteriorhodopsin (17 A) and consistent with current ideas about the location of retinal in the interior of the protein. In the absence of energy transfer (bleached vesicles), the steady-state fluorescence anisotropy, -r, of DPH is considerably lower than in the corresponding unbleached vesicles, indicating that the effect of energy transfer must be taken into account when interpreting -r in terms of order and dynamics.     

Effect of aminophospholipid glycation on order parameter and hydration of phospholipid bilayer

The effect of aminophospholipid glycation on lipid order and lipid bilayer hydration was investigated using time-resolved fluorescence spectroscopy. The changes of lipid bilayer hydration were estimated both from its effect on the fluorescence lifetime of The 1-[4-(trimethylammonium)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and 1,6-diphenylhexa-1,3,5-triene (DPH) and using solvatochromic shift studies with 1-anilinonaphthalene-8-sulfonic acid. The head-group and acyl chain order were determined from time-resolved fluorescence anisotropy measurements of the TMA-DPH and DPH. The suspensions of small unilamellar vesicles (with phosphatidylethanolamine/phosphatidylcholine molar ratio 1:2.33) were incubated with glyceraldehyde and it was found that aminophospholipids react with glyceraldehyde to form products with the absorbance and the fluorescence properties typical for protein advanced glycation end products. The lipid glycation was accompanied by the progressive oxidative modification of unsaturated fatty acid residues. It was found that aminophospholipid glycation increased the head-group hydration and lipid order in both regions of the membrane. The lipid oxidation accompanying the lipid glycation affected mainly the lipid order, while the effect on the lipid hydration was small. The increase in the lipid order was presumably the result of two effects: (1) the modification of head-groups of phosphatidylethanolamine by glycation; and (2) the degradation of unsaturated fatty acid residues by oxidation.     

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.     

Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations.

Differential polarized phase fluorometry has been used to investigate the depolarizing rotations of 1,6-diphenyl-1,3,5-hexatriene (DPH) in isotropic solvents and in lipid bilayers. For DPH dissolved in isotropic solvents, there is a precise agreement between the observed and predicted values for maximum differential tangents, indicating that in these media DPH is a free isotropic rotator. In lipid bilayers the tangent defects (i.e., the differences between the calculated and the observed maximum differential tangents) are too large to be explained by anisotropy in the depolarizing rotations but are accounted for by hindered isotropic torsional motions for the fluorophore [Weber, G (1978) Acta Phys. Pol A 54, 173]. This theory describes the depolarizing rotations of the fluorophore by its rotational rate R (in radians/second) and the limiting fluorescence anisotropy (r) at times long compared with the fluorescence lifetime. Through the combined use of both steady-state anisotropy measurements and differential phase measurements, we have demonstrated that one may obtain unique solutions for both R and r. For DPH embedded in vesicles prepared from dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholines, the depolarizing motions are highly hindered at temperatures below the transition temperature (Tc) but are unhindered above Tc. The apparent rotational rates of the probe do not change significantly at Tc. These data suggest that the changes observed in the steady-state anisotropy near Tc derive primarily from changes in the degree to which the probe's rotations are hindered, and only to a small extent from changes in rotational rate. For DPH embedded in bilayers that contained 25 mol % cholesterol, no clear transition occurred and the rotations appeared to be hindered at all temperatures. The rotational motions of DPH embedded in dioleolyphosphatidylcholine were found to be far less hindered, but the rotational rates were similar to those obtained in the saturated phosphatidylcholines. Finally, the data show that in an anisotropic environment, such as that of a lipid bilayer, steady-state fluorescence anisotropy measurements alone cannot yield quantitatively meaningful rotational rates. Extrapolation of steady-state aniosotropy data to the quantitation of membrane viscosity is therefore difficult, if not invalid; however, qualitative comparisons can be useful.     

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.     

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles

The fluorescence emission properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dipalmitoyl-3-sn-phosphatidylcholine and 1,2-dimyristoyl-3-sn-phosphatidylcholine multilamellar vesicles have been measured by using multifrequency phase fluorometry. The fluorescence decay of DPH in the phospholipid vesicles has been analyzed by assuming either that the decay is made up of a discrete sum of exponential components or that the decay is made up of one or more continuous distributions of lifetime components. The fit of the decay curve using exponentials required at least two terms, and the reduced X2 was relatively large. The fit using a continuous distribution of lifetime values used two continuous components. Several symmetric distribution functions were used: uniform, Gaussian, and Lorentzian. The distribution function that best described the decay was the Lorentzian. The full width at half-maximum of the Lorentzian distribution was about 0.6 ns at temperatures below the phase transition temperature. At the phospholipid phase transition and at higher temperatures, the distribution became quite narrow, with a width of about 0.1 ns. It is proposed that the lifetime distribution is generated by a continuum of different environments of the DPH molecule characterized by different dielectric constants. Below the transition temperature in the gel phase, the dielectric constant gradient along the membrane normal determines the distribution of decay rates. Above the transition, in the liquid-crystalline phase, the translational and rotational mobility of the DPH molecule increases, and the DPH experiences an average environment during the excited-state lifetime. Consequently, the distribution becomes narrower.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Physical characterization of cyclosporine binding sites in lymphocytes.

The binding of cyclosporine to human peripheral blood lymphocytes (PBLs) was studied by measuring the fluorescence emission spectrum and lifetime of the fluorescent and immunosuppressive cyclosporine derivative dansyl-cyclosporine (DCs). The emission maximum and fluorescence lifetime of DCs were characterized in several solvents. The fluorescence emission maximum and lifetime of DCs increased at a high dielectric constant. The fluorescence lifetime decay curve of DCs was a monoexponential function in all solvents tested. Fluorescence micrographs of lipid vesicles and erythrocytes labeled with DCs exhibit uniform staining patterns, whereas PBLs show heterogeneous DCs labeling. DCs exhibits a relatively low emission maximum (490 nm) in erythrocyte membranes. Such an emission maximum is characteristic of a hydrophobic environment. DCs in PBLs also has a low emission maximum (484 nm). The lifetime of DCs in PBLs required two exponential terms to properly fit the lifetime decay curve and could not be attributed to light scattering. One short component (4.7 +/- 1.0 ns) and a second long component (18.5 +/- 1.0 ns) were resolved from the DCs fluorescence decay curves. Time-resolved anisotropy of DCs in PBLs revealed that the labeled drug was present in an anisotropic environment, consistent with at least some DCs being bound to a membrane. These fluorescence studies suggest that DCs interacts with multiple and/or heterogeneous sites in peripheral blood lymphocytes.     

Large solutes induce structural perturbations in proteins and membranes.

Structural perturbations in biopolymers with hydrophobic interiors i.e. specific proteins and dimyristoylphosphatidylcholine (DMPC) vesicles were investigated as a function of solute concentrations in the medium. 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as fluorescent probe. Response of DPH was comparable to that of intrinsic tryptophan in BSA in terms of steady state and time resolved fluorescence. The solutes induced a decrease in steady state anisotropy as well as rotational correlation time (computed from lifetime measurements) for DPH in both proteins and membranes. Enhanced access of the quencher potassium iodide to tryptophan in bovine serum albumin (BSA) and ovalbumin, and enhanced terbium leakage in DMPC vesicles induced by various solutes concomitant with decreased anisotropy/correlation time were consistent with structural perturbations of the nature of defects or voids in these polymers.     

Combining fluorescence lifetime and polarization microscopy to discriminate phase separated domains in giant unilamellar vesicles

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42 mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦(Triticum sativum L.)根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯(DPH)荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H     

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.     

Modulation of pig kidney Na+/K+-ATPase activity by cholesterol: role of hydration

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.     

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.     

Gangliosides asymmetrically alter the membrane order in cultured PC-12 cells

Exogenous gangliosides readily associate with the cell membranes and produce marked effects on cell growth and differentiation. We have studied the effect of bovine brain gangliosides (BBG) on the membrane dynamics of intact cells. The structural and dynamic changes in the cell membrane were monitored by the fluorescence probes DPH, TMA-DPH and laurdan. Incorporation of BBG into the cell membrane decreased the fluorescence intensity, lifetime and the steady state anisotropy of TMA-DPH. Analysis of the time resolved anisotropy decay by wobbling in the cone model revealed that BBG decreased the order parameter, and increased the cone angle without altering the rotational relaxation rate. The fluorescence intensity and lifetime of DPH were unaffected by BBG incorporation, however, a modest increase was observed in the steady state anisotropy. BBG incorporation reduced the total fluorescence intensity of laurdan with pronounced quenching of the 440-nm band. The wavelength sensitivity of generalized polarization of laurdan manifested an ordered liquid crystalline environment of the probe in the cell membrane. BBG incorporation reduced the GP values and augmented the liquid crystalline behavior of the cell membrane. BBG incorporation also influenced the permeability of cell membranes to cations. An influx of Na+ and Ca2+ and an efflux of K+ was observed. The data demonstrate that incorporation of gangliosides into the cell membrane substantially enhances the disorder and hydration of the lipid bilayer region near the exoplasmic surface. The inner core region near the center of the bilayer becomes slightly more ordered and remains highly hydrophobic. Such changes in the structure and dynamics of the membrane could play an important role in modulation of transmembrane signaling events by the gangliosides.     

Fluorescent Probes DPH,TMA-DPH and C17-HC Induce Erythrocyte Exovesiculation

An experimental approach has been developed to study human erythrocyte vesiculation, using the fluorescent probes diphenylhexatriene (DPH), trimethylamino-diphenylhexatriene (TMA-DPH) and heptadecyl-hydroxycoumarin (C17-HC). Acetylcholinesterase (AChE) enzyme activity measurements confirmed the presence of exovesicles released from erythrocyte membranes labeled with DPH, TMA-DPH or C17-HC. The fluorescence intensity and anisotropy values obtained showed that the amphiphilic probes TMA-DPH and C17-HC are preferentially incorporated in the exovesicles (when compared with DPH). There is a significant decrease of the cholesterol content of the exovesicle suspensions with time, independently of the fluorescence probe used, reaching undetectable cholesterol levels for the samples incubated for 48 hr. The ratios between the concentration of cholesterol released in the exovesicles after 1 hr incubation with DPH, TMA-DPH or C17-HC and the probe concentration used in the incubation were 84.7, 3.82 and 0.074, respectively. The size of the released vesicles was evaluated by dynamic light scattering spectroscopy. Some hypotheses are proposed that could explain the resemblance and differences between the results obtained for erythrocytes labeled with each probe, considering the present knowledge of membrane vesiculation mechanisms, lipid microdomains (rafts), erythrocyte membrane phospholipid asymmetry and AChE inhibition by TMA-DPH and C17-HC. This work demonstrates that the fluorescent probes DPH, TMA-DPH and C17-HC induce rapid erythrocyte exovesiculation; their use can lead to new methodologies for the study of this still poorly understood mechanism.