Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes

Understanding the mechanism that governs the transport of long chain free fatty acids (FFA) across lipid bilayers is critical for understanding transport across cell membranes. Conflicting results have been reported for lipid vesicles; most investigators report that flip-flop occurs within the resolution time of the method (<5 ms) and that dissociation from the membrane is rate limiting, while other studies find that flip-flop is rate limiting and on the order of seconds. We have reinvestigated this problem and find that the methods used in studies reporting rapid flip-flop have not been interpreted correctly. We find that accurate information about transport of FFA across lipid vesicles requires that FFA be delivered to the vesicles as complexes with albumin (BSA). For example, we find that stopped-flow mixing of uncomplexed FFA with small unilamellar vesicles (SUV) containing pyranine yields the very fast influx rates reported previously (>100 s(-1)). However, these influx rates increase linearly with lipid vesicle concentration and can therefore not, as previously interpreted, represent flip-flop. In contrast, measurements of influx rates in SUV and giant unilamellar vesicles performed with oleate-BSA complexes reveal no dependence on vesicle concentration and yield influx rate constants of approximately 4 and approximately 0.5 s(-1), respectively. Rate constants for efflux and dissociation were determined from the transfer of oleate from vesicles to BSA and reveal similar influx and efflux but dissociation rate constants that are approximately 5-10-fold greater. We conclude that flip-flop is rate limiting for transport of FFA across lipid vesicles and slows with an increasing radius of curvature. These results, in contrast to those reporting that flip-flop is extremely fast, indicate that the lipid bilayer portion of biological membranes may present a significant barrier to transport of FFA across cell membranes.     

Mechanisms of cellular uptake of long chain free fatty acids

Cells take up long chain free fatty acids (FFA) in vivo from the non-protein bound ligand pools in extracellular fluid and plasma, which contain ~100 and 600 M albumin, respectively. The physiologic range of unbound FFA concentrations in such fluids has traditionally been calculated at < 1 M. Studies of [3H]-oleate uptake by hepatocytes, adipocytes, cardiac myocytes and other cell types demonstrate that FFA uptake within this range is saturable, and exhibits many other kinetic properties indicative of facilitated transport. Within this range, the uptake kinetics of the acidic (pKa = 0.5) FFA analog 2,2,3- heptafluorostearate are similar to those of stearate. Thus, uptake of physiologic concentrations of FFA involves facilitated transport of the FFA anion (FA-). Over a much wider range of unbound FFA concentrations hepatocellular [3H]-oleate uptake exhibits both saturable and non-saturable components. Oleate binding to liver plasma membranes (LPM) also demonstrates such components. Comparing the two components of FFA uptake to the corresponding components of binding permits estimates of trans-membrane transport rates. T1/2 for saturable uptake (~ 1 sec) is less than for non-saturable uptake (~ 14 sec). Others have determined the flip-flop rates of protonated FFA (FAH) across small and large unilamellar vesicles (SUV, LUV) and across cellular plasma membranes. These reported flip-flop rates, measured by the decrease in pH resulting from the accompanying proton flux, exhibit a highly significant inverse correlation with cell and vesicle diameter (r = 0.99). Although T1/2's in vesicles are in the msec range, those in cells are > 10 sec, and thus comparable to the rates of non-saturable uptake we determined. Thus, under physiologic conditions, the predominant mechanism of cellular FFA uptake is facilitated transport of FA-; at much higher, non- physiologic FFA concentrations, passive flip-flop of FAH predominates. Several plasma membrane proteins have been identified as potential mediators of facilitated FFA transport. Studies in animal models of obesity and non-insulin dependent diabetes mellitus demonstrate that tissue-specific regulation of facilitated FFA transport has important pathophysiologic consequences.     

Exchange and flip-flop of dimyristoylphosphatidylcholine in liquid-crystalline, gel, and two-component, two-phase large unilamellar vesicles

The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrasonic absorption and permeability for liposomes near phase transition

The specific ultrasonic absorption coefficient per wavelength as a function of temperature in the vicinity of the phase transition of liposomes, composed of a 4:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), of different sizes was determined using an acoustic interferometer. Small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) yielded results similar to those in the literature, viz., an absorption maximum at the transition temperature. Seven intermediate sizes including several size distributions of large unilamellar vesicles (LUV) were studied, yielding information on size dependencies of the temperatures at which the peaks occur, the widths at half peak amplitude, and the peak amplitudes. All liposome sizes except the SUV exhibited approximately the same transition temperature as did the largest MLV. The widths of the peaks were inversely related to liposome size, with a strong dependence for the smallest vesicles and an approach to independence for the largest vesicles. The amplitudes of the peaks exhibited a general increase with size with two exceptions, viz., the SUV and the vesicles with average diameters of 90-100 nm. It was also found that the membrane permeability increased near the transition temperature. The temperature dependencies of ultrasonic absorption and membrane permeability are compared.     

Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.     

Temperature dependence of anion transport inhibitor binding to human red cell membranes

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30 degrees C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 +/- 0.9 kcal/mol and 3.2 +/- 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 +/- 0.9 kcal/mol (bimolecular, forward step), 17 +/- 2 kcal/mol (bimolecular, reverse step), 6.4 +/- 2.3 kcal/mol (unimolecular, forward step), and 10.6 +/- 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.     

Factors contributing to the distribution of free fatty acids among phospholipid vesicles

The distribution of free fatty acids at equilibrium after incubation of small sonicated unilamellar vesicles (SUV) with large unilamellar vesicles (LUV) of different lipid composition has been determined. Stearic acid (SA) and oleic acid (OA) showed similar preferences for SUV and LUV of egg yolk phosphatidylcholine (EYPC). Both ionized and protonated forms of the free fatty acids (FFAs) behaved similarly with respect to the equilibrium distribution between EYPC of different size. The charge of the vesicles was found, however, to be important, since both FFAs in their ionized form preferentially associated to vesicles of phosphatidylcholine (PC) as compared with vesicles of phosphatidylglycerol (PC). While SA preferred membranes in the gel state, OA showed preference for the membrane in fluid state. The insertion of both OA and SA in phosphatidylethanolamine (PE)/phosphatidylcholine vesicles is less favourable than in vesicles of pure PC. All these data suggest that membrane lipid content may play a role in determining the distribution of free fatty acids among the membranes of a cell.     

Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.     

Thermodynamics and kinetics of phospholipid monomer-vesicle interaction

Resonance energy transfer between acyl chain labeled (7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylcholine (NBD-PC) and head group labeled (lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) was used to monitor the rate of NBD-PC transfer between two populations of dioleoylphosphatidylcholine (DOPC) vesicles. Equilibration of NBD-PC between DOPC vesicles occurs by the diffusion of soluble monomers through the water phase, which is a first-order process. Conditions were used such that the apparent transfer rate constant is equal to the rate constant for monomer-vesicle dissociation into solution. The partition distribution of NBD-PC between DOPC vesicles and water was determined by measuring the loss of NBD-PC from vesicles into solution following the dilution of small amounts of vesicles in buffer. The acyl chain length and temperature dependence of both the rate and partition measurements were determined, and a free energy diagram for NBD-PC-soluble monomer-vesicle interactions was constructed. The conclusions of this analysis are the following: NBD-PC dissociation from and association with the bilayer require passage through a high-energy transition state resulting predominantly from enthalpic energy. The activation energy for NBD-PC-vesicle dissociation becomes more positive and the standard free energy of NBD-PC transfer from water to vesicles becomes more negative with increasing acyl chain length. The standard free energy of transfer for NBD-PC from water to vesicles results predominantly from differences in enthalpy between the membrane and water phases. The enthalpy of activation for association increases with acyl chain length and is larger than expected for an aqueous diffusion-limited process in bulk water.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.     

Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles

We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free fatty acids modulate intermembrane trafficking of cholesterol by increasing lipid mobilities: novel 13C NMR analyses of free cholesterol partitioning

Cholesterol and free fatty acids in membranes modulate major biological processes, and their cellular metabolism and actions are often coordinately regulated. However, effects of free fatty acid on cholesterol-membrane interactions have proven difficult to monitor in real time in intact systems. We developed a novel (13)C NMR method to assess effects of free fatty acids on molecular interactions of cholesterol within--and transfer between--model membranes. An important advantage of this method is the ability to acquire kinetic data without separation of donor and acceptor membranes. Large unilamellar phospholipid vesicles (LUV) with phosphatidylcholine/cholesterol ratios of 4:1 served as cholesterol donors. Small unilamellar vesicles (SUV) made with phosphatidylcholine were acceptors. The (13)C(4)-cholesterol peak is narrow in SUV, but very broad in LUV, spectra; the increase in intensity of this peak over time monitored transfer. Oleic acid and other long chain free fatty acids [saturated (C12-18) and unsaturated (C18)] dose-dependently increased mobilities of lipids in LUV (phospholipid and cholesterol) and cholesterol transfer rates, whereas short (C8-10) and very long (C24) chain free fatty acids did not. Decreasing pH from 7.4 to 6.5 (+/-oleic acid) had no effect on cholesterol transfer, and 5 mol % fatty acyl-CoAs increased transfer rates, demonstrating greater importance of the fatty-acyl tail over the headgroup. In LUV containing sphingomyelin, transfer rates decreased, but the presence of oleic acid increased transfer 1.3-fold. These results demonstrate free fatty acid-facilitated cholesterol movement within and between membranes, which may contribute to their multiple biological effects.     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.     

Fusogenic capacities of divalent cations and effect of liposome size

The initial kinetics of divalent cation (Ca2+, Ba2+, Sr2+) induced fusion of phosphatidylserine (PS) liposomes, LUV, is examined to obtain the fusion rate constant, f11, for two apposed liposomes as a function of bound divalent cation. The aggregation of dimers is rendered very rapid by having Mg2+ in the electrolyte, so that their subsequent fusion is rate limiting to the overall reaction. In this way the fusion kinetics are observed directly. The bound Mg2+, which by itself is unable to induce the PS LUV to fuse, is shown to affect only the aggregation kinetics when the other divalent cations are present. There is a threshold amount of bound divalent cation below which the fusion rate constant f11 is small and above which it rapidly increases with bound divalent cation. These threshold amounts increase in the sequence Ca2+ less than Ba2+ less than Sr2+, which is the same as found previously for sonicated PS liposomes, SUV. While Mg2+ cannot induce fusion of the LUV and much more bound Sr2+ is required to reach the fusion threshold, for Ca2+ and Ba2+ the threshold is the same for PS SUV and LUV. The fusion rate constant for PS liposomes clearly depends upon the amount and identity of bound divalent cation and the size of the liposomes. However, for Ca2+ and Ba2+, this size dependence manifests itself only in the rate of increase of f11 with bound divalent cation, rather than in any greater intrinsic instability of the PS SUV. The destabilization of PS LUV by Mn2+ and Ni2+ is shown to be qualitatively distinct from that induced by the alkaline earth metals.     

How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?

Tryptophan fluorescence is a powerful tool for studying protein structure and function, especially membrane-active proteins and peptides. It is arguably the most frequently used tool for examining the interactions of proteins and peptides with vesicular unilamellar model membranes. However, high light scattering associated with vesicular membrane systems presents special challenges. Because of their reduced light scattering compared to large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) produced by sonication are widely used membrane models. Unfortunately, SUV, unlike LUV, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. We present simple and easily implemented experimental procedures for the accurate determination of tryptophan (Trp) fluorescence in either LUV or SUV. Specifically, we show that Trp spectra can be obtained in the presence of up to 6 mM LUV that are virtually identical to spectra obtained in buffer alone, which obviates the use of SUV. We show how the widths and peak positions of such spectra can be used to evaluate the heterogeneity of the membrane conformation and penetration of peptides. Finally, we show how to use a reference fluorophore for the correction of intensity measurements so that the energetics of peptide partitioning into membranes can be accurately determined.     

The effect of liposome size on the final lipid/DNA ratio of cationic lipoplexes

Several studies have demonstrated that lipoplexes are two-phase systems over most mixing lipid/DNA charge ratios. Because these studies have focused on small unilamellar vesicles (SUV), they leave open the question as to whether a similar pattern is followed by other liposome types. The main purpose of this work is to examine the question further by characterizing the assembly of cationic lipoplexes prepared from 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM)/dioleoylphosphatidylethanolamine (DOPE) (1:1) liposomes of various types. Sedimentation in sucrose density gradients reveals that large unilamellar vesicles (LUV) and sedimented multilamellar vesicles (sMLV), as opposed to SUV, form lipoplexes that exist as a single phase over a relatively broad range of mixing (+/-) ratios. This is indicated by observing that most of the LUV and sMLV become involved in the assembly reaction up to mixing (+/-) ratios of 4 and 9, respectively, while only a small and constant fraction of SUV associates with DNA at all mixing (+/-) ratios tested. Consequently, while maximal (+/-) ratios of approximately 4.5 and 9 are found in LUV and sMLV lipoplexes, respectively, a final (+/-) ratio of only approximately 2 is determined in SUV lipoplexes. Isothermal titration calorimetry shows that this is the lowest possible charge ratio achieved when liposomes are titrated with DNA. Based on these observations and on the size differences of the liposomes used, a model of lipoplex formation is proposed.     

Preparation and characterization of monodisperse unilamellar phospholipid vesicles with selected diameters of from 300 to 600 nm

A method has been developed for making large unilamellar vesicles (LUV) with low polydispersity. The LUV, constituted of dioleoylphosphatidic acid (DOPA), 300 nm in diameter are made by a modification of the pH adjustment technique (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683-1687). This size is 10 times that (30 nm) of vesicles prepared by prolonged sonication. Vesicle size is increased stepwise by adding cholesterol (to a maximum of 40 mol% cholesterol) to form vesicles in 0.15 M KCl with up to 600 nm diameter. The vesicle size is measured by photon correlation spectroscopy, electron microscopy, and by measurement of the internal volume with cyanocobalamin while calculating the number of DOPA molecules per vesicle. Vesicles are stable for at least three weeks. Sepharose 4B column chromatography of the preparation yields a peak of fractions with the same polydispersity as the original sample and shows that 30 to 40% of the original lipid in a sample is recovered as LUV. Less than 2% of the sample forms small unilamellar vesicles (SUV) (diameter = 30 nm), which emerge from the column in a separate peak. Since the remaining lipid is not suspended in the buffer during vesicle formation, for most purposes the vesicles may be used immediately after titration so that they can be prepared in less than 40 min.     

Dioxygen transmembrane distributions and partitioning thermodynamics in lipid bilayers and micelles

Cellular respiration, mediated by the passive diffusion of oxygen across lipid membranes, is key to many basic cellular processes. In this work, we report the detailed distribution of oxygen across lipid bilayers and examine the thermodynamics of oxygen partitioning via NMR studies of lipids in a small unilamellar vesicle (SUV) morphology. Dissolved oxygen gives rise to paramagnetic chemical shift perturbations and relaxation rate enhancements, both of which report on local oxygen concentration. From SUVs containing the phospholipid sn-2-perdeuterio-1-myristelaidoyl, 2-myristoyl-sn-glycero-3-phosphocholine (MLMPC), an analogue of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), we deduced the complete trans-bilayer oxygen distribution by measuring (13)C paramagnetic chemical shifts perturbations for 18 different sites on MLMPC arising from oxygen at a partial pressure of 30 bar. The overall oxygen solubility at 45 °C spans a factor of 7 between the bulk water (23.7 mM) and the bilayer center (170 mM) and is lowest in the vicinity of the phosphocholine headgroup, suggesting that oxygen diffusion across the glycerol backbone should be the rate-limiting step in diffusion-mediated passive transport of oxygen across the lipid bilayer. Lowering of the temperature from 45 to 25 °C gave rise to a slight decrease of the oxygen solubility within the hydrocarbon interior of the membrane. An analysis of the temperature dependence of the oxygen solubility profile, as measured by (1)H paramagnetic relaxation rate enhancements, reveals that oxygen partitioning into the bilayer is entropically favored (ΔS° = 54 ± 3 J K(-1) mol(-1)) and must overcome an enthalpic barrier (ΔH° = 12.0 ± 0.9 kJ mol(-1)).