States of aggregation and phase transformations in mixtures of phosphatidylcholine and octyl glucoside

The result of mixing varying concentrations of the nonionic detergent octyl glucoside (OG) with small unilamellar vesicles (SUV) of egg phosphatidylcholine (PC) made by sonication depends on the ratio between OG and PC in the mixed aggregates. When this molar ratio (Re) is lower than 1.4, the detergent partitions between the PC vesicles and the aqueous medium with a partition coefficient of K = 0.033 mM-1. As a consequence of introduction of OG into the bilayers, the vesicles grow in size. The resultant vesicles have a mean diameter that is an increasing function of Re and is independent of the total PC concentration. Experiments in which the vesicles were loaded with high molecular weight dextran prior to being exposed to OG suggest that the mechanism responsible for the size growth involves lipid transfer rather than fusion. Mixtures with Re values within the range of 1.4-3.2 separate into two macroscopic phases: The lower phase is clear but very viscous. It contains constant OG and PC concentrations and is characterized by an Re value of 3.2, independent of the composition of the whole dispersion. The upper phase contains vesicles of varying concentrations of OG and PC, but a constant Re of 1.4. When the saturating level of 1.4 OG molecules per PC molecule is approached, the concentration of OG monomers in the aqueous medium reaches the value of 16.6 +/- 0.3 mM, which is the apparent cmc of OG in the lipid-containing medium. OG-PC mixed micelles contain at least 3.2 OG molecules per PC molecule. The mixed micelles present at Re = 3.2 apparently have the shape of oblate ellipsoids with a minor axis of about 2 nm and two major axes of about 25 nm. The surface area of the mixed micelles at this point is just sufficient for them to undergo conversion into the smallest possible spherical vesicles of a radius of 12 nm. At Re values above 3.2, the major axis of the mixed micelles becomes smaller as Re increases, while at values of Re below 3.2 the micelles would have been expected to grow very rapidly with decreasing Re. This may explain the partial vesicle closure occurring below Re = 3.2.     

Temperature dependence of the vesicle-micelle transition of egg phosphatidylcholine and octyl glucoside

The temperature dependence of octyl glucoside micellization was determined and compared to the phase behavior of the octyl glucoside--egg phosphatidylcholine (PC) mixed system in excess water to help elucidate the process of vesicle formation from mixed surfactant-phospholipid micelles. The critical micelle concentration of octyl glucoside (OG) was determined from the sharp increase of ANS fluorescence at micellization in an NaCl buffer at temperatures ranging from 5 to 40 degrees C. The cmc decreased with increasing temperature from 31 mM at 5 degrees C to 16 mM at 40 degrees C. A similar but less steep temperature dependence is observed for the solubilization of egg PC vesicles by OG as monitored by the surfactant-dependent changes in (1) solution turbidity and (2) the resonance energy transfer between NBD-PE and Rho-PE incorporated in the vesicles. These assays identify two breakpoints, most likely the boundaries of the cylindrical micelle and spheroidal micelle coexistence region. The [OG]aq values at these two breakpoints have similar temperature dependencies. However, the cylindrical mixed micelles at the boundary have nearly identical OG:PC ratios over the temperature range studied, whereas the spheroidal mixed micelles have relatively more OG at the higher temperatures (OG:PC ratio increases from 2.92 to 3.72 between 5 and 35 degrees C). Estimation of the acyl volume to surface area ratio for the compositions observed suggests that this parameter remains constant over temperature. The spheroidal mixed micelles, but not the cylindrical PC-OG micelles, exhibit ideal mixing between the two components at all temperatures (5-35 degrees C). This temperature sensitivity may be utilized to improve the efficacy of membrane protein reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of interaction of octyl glucoside with phosphatidylcholine vesicles: partitioning and solubilization as studied by high sensitivity titration calorimetry

The interaction of the surfactant octyl glucoside (OG) with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and soy bean phosphatidylcholine (soy bean PC) was studied using high-sensitivity titration calorimetry. We determined the partition coefficient of OG between water and lipid bilayers and the transfer enthalpy of the surfactant by addition of lipid vesicles to OG monomers or vice versa. Comparison with the micellization enthalpy of the surfactant gives information on differences in the hydrophobic environment of OG in a liquid-crystalline bilayer or a micelle. The average partition coefficient P in mole fraction units for x_e≈0.12–0.2 decreases slightly from 4152 at 27°C to 3479 at 70°C for DMPC and from 4260 to 3879 for soy bean PC, respectively. The transfer enthalpy ΔH^T of OG into lipid vesicles is positive at 27°C and negative at 70°C. Its temperature dependence is larger for the incorporation of OG into DMPC than into soy bean PC vesicles. It is concluded that OG in DMPC vesicles is better shielded from water than in soy bean PC vesicles or in micelles. Titration calorimetry was also used to determine the phase boundaries of the coexistence region of mixed vesicles and mixed micelles in the systems OG/DMPC, OG/DPPC, OG/DSPC, and OG/soy bean PC vesicles at 70°C in the liquid-crystalline phase. DMPC and soy bean PC solubilization was also studied at 27°C to investigate the effect of temperature. The effective surfactant to lipid ratios at saturation, R_e^sat, for all PCs studied are in the range between 1.33–1.72 and the ratios at complete solubilization, R_e^sol, are between 1.79–3.06. At 70°C, the R_e^sat values decrease with increasing chain length of the saturated PC. The ratios depend also slightly on temperature and the degree of unsaturation of the fatty acyl chains. For the OG/soy bean PC system, the coexistence range for mixed vesicles and mixed micelles is larger than for the corresponding PCs with saturated chains.     

Vesicle-micelle transition of phosphatidylcholine and octyl glucoside elucidated by cryo-transmission electron microscopy.

Vesicle-micelle transition structures of egg phosphatidylcholine (PC) and octyl glucoside (OG) mixtures were observed in the vitrified hydrated state by cryo-transmission electron microscopy (cryo-TEM) and correlated with the macroscopic and molecular changes previously associated with micellization monitored by 90 degrees light scattering and resonance energy transfer between fluorescent lipid probes. Several distinct structural changes occurred as OG was added to the PC vesicles. First, the average vesicle size decreased from 160 nm to less than 66 nm with no apparent change or decrease in optical density (OD). Then, associated with a small rise in OD, samples with open vesicles were observed coexisting with pieces of lamellae and long cylindrical micelles; more micelles were seen at higher [OG]. This mixture of vesicles and cylindrical micelles occurred in the region of the phase diagram previously attributed to vesicle opening, and possibly vesicle size increase. At higher [OG], small spheroidal micelles coexisting with cylindrical micelles correlated with a decrease in OD and changes in the fluorescence signal. At high [OG] when the solution appeared clear, spheroidal micelles were the dominant structure. By using cryo-TEM, a technique which preserves the original microstructure of fluid systems and provides direct images at 1 nm resolution, we have elucidated the vesicle-micelle transition and identified intermediates not known previously in the PC/OG system.     

The heat of transfer of lipid and surfactant from vesicles into micelles in mixtures of phospholipid and surfactant.

We study the heat associated with the transformation of vesicles into micelles in mixtures of bilayer-forming phospholipids and micelle-forming surfactants. We subdivide the total heat evolution deltaQ(coex) within the range of coexistence of vesicles and micelles into three contributions related to the transition of dN(D)m-b molecules of surfactant and dN(L)m-b molecules of lipid from micelles to vesicles and to the extraction of dN(D)m-w molecules of surfactant from micelles to the aqueous solution, so that deltaQ(coex) = deltaH(D)m-w x dN(D)m-w + deltaH(D)m-b x dN(D)m-b + deltaH(L)m-b x dN(L)m-b where deltaH(D)m-w, deltaH(L)m-b, and deltaH(D)m-b are the respective molar "transfer" enthalpies. We design a method for the evaluation of all three molar enthalpies, from isothermal calorimetric titrations conducted according to two different protocols of titration of lipid-surfactant mixtures. In the first protocol the mixture is titrated with an aqueous solution of pure lipid vesicles, and in the second the mixture is titrated with an aqueous solution of pure surfactant. Titration of the mixed systems by a buffer solution serves to verify the results obtained under these protocols. In addition to the values of molar enthalpies, our method yields the cmc value of the pure surfactant. We apply our method to investigating the heat evolution in mixtures of egg yolk phosphatidylcholine and the nonionic surfactant octylglucoside in a phosphate-buffered saline solution at 28 degrees C. These studies gave the following values: deltaH(D)m-w = -1732 cal/mol, deltaH(L)m-b = -592 cal/mol, deltaH(D)m-b = 645 cal/mol, and cmc = 23.5 mM. We discuss the possible physical insight of these values and the perspectives of applications of the proposed method.     

Partition coefficient of a surfactant between aggregates and solution: application to the micelle-vesicle transition of egg phosphatidylcholine and octyl beta-D-glucopyranoside.

The mechanism of the solubilization of egg phosphatidylcholine containing 10% (M/M) of egg phosphatidic acid unilamellar vesicles by the nonionic detergent, octyl beta-D-glucopyranoside, has been investigated at both molecular and supramolecular levels by using fluorescence and turbidity measurements. In the lamellar region of the transition, the solubilization process has been shown to be first a function of the initial size before reaching an equilibrium aggregation state at the end of this region (the onset of the micellization process). The analysis during the solubilization process of the evolution of both the fluorescence energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (Rho-PE) and the fluorescence of 6-dodecanoyl-2-dimethylaminoaphtalene (Laurdan) has allowed us to determine the evolution of the detergent partitioning between the aqueous and the lipidic phases, i.e., the evolution of the molar fraction of OG in the aggregates (XOG/Lip) with its monomeric detergent concentration in equilibrium ([OG]H2O), throughout the vesicle-to-micelle transition without isolating the aqueous medium from the aggregates. The curve described by XOG/Lip versus [OG]H2O shows that the partition coefficient of OG is changing throughout the solubilization process. From this curve, which tends to a value of 1/(critical micellar concentration), five different domains have been delimited: two in the lamellar part of the transition (for 0 < [OG]H2O < 15.6 mM), one in the micellization part, and finally two in the pure micellar region (for 16.5 < [OG]H2O < 21 mM). The first domain in the lamellar part of the transition is characterized by a continuous variation of the partition coefficient. In the second domain, a linear relation relates XOG/Lip and [OG]H2O, indicating the existence of a biphasic domain for which the detergent presents a constant partition coefficient of 18.2 M-1. From the onset to the end of the solubilization process (domain 3), the evolution of (XOG/Lip) with [OG]H2O can be fitted by a model corresponding to the coexistence of detergent-saturated lamellar phase with lipid-saturated mixed micelles, both in equilibrium with an aqueous phase, i.e., a three-phase domain. The micellar region is characterized first by a small two-phase domain (domain 4) with a constant partition coefficient of 21 M-1, followed by a one-phase mixed-micellar domain for which XOG/Lip no longer linearly depends on [OG]H2O. The results are discussed in terms of a phase diagram.     

Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside

The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of the micelle-vesicle transition in lecithin-bile salt solutions.

Small-angle neutron scattering (SANS) and dynamic light scattering (QLS) are used to characterize the aggregates found upon dilution of mixed lecithin-bile salt micelles. Molar ratios of lecithin (L) to taurocholate (TC) studied varied from 0.1 to 1, and one series contained cholesterol (Ch). Mixed aggregates of L and taurodeoxycholate (TDC) at ratios of 0.4 and 1 were also examined. In all cases the micelles are cylindrical or globular and elongate upon dilution. The radius of the mixed micelles varies only slightly with the overall composition of lecithin and bile salt which indicates that the composition of the cylindrical micelle body is nearly constant. The transition from micelles to vesicles is a smooth transformation involving a region where micelles and vesicles coexist. SANS measurements are more sensitive to the presence of two aggregate populations than QLS. Beyond the coexistence region the vesicle size and degree of polydispersity decrease with dilution. Incorporation of a small amount of cholesterol in the lipid mixture does not affect the sequence of observed aggregate structures.     

Kinetic and structural aspects of reconstitution of phosphatidylcholine vesicles by dilution of phosphatidylcholine-sodium cholate mixed micelles

Dilution of mixed micellar dispersions of egg phosphatidylcholine (PC) and sodium cholate beyond a critical value results in formation of cholate-containing PC vesicles. The structure of the resultant vesicles and some mechanistic aspects of this process have been investigated by the use of light scattering and nuclear magnetic resonance techniques. The main findings and conclusions are the following: Both the state of aggregation (micellar or vesicular) and the apparent equilibrium size distribution of micelles or vesicles obtained by dilution of the PC-cholate mixed micellar dispersions are a function of the cholate to PC molar ratio in the mixed aggregates (micelles or vesicles). When this effective ratio (Re) is higher than 0.4, the dispersion is micellar, and the size of the mixed micelles increases with decreasing Re; when Re less than 0.3, the dispersion is essentially vesicular, and the mean hydrodynamic radius of the vesicles is an increasing function of Re; in dispersions with 0.3 less than Re less than 0.4, mixed micelles and vesicles coexist. Addition of cholate to vesicular dispersions, to Re values below 0.3, results in vesicle size growth through a concentration-independent lipid-exchange mechanism. Addition of cholate to higher Re values results in micellization (solubilization) of the vesicles. On the other hand, dilution of vesicular dispersions does not affect the size of the vesicles. Apparent equilibration of a mixed micellar dispersion following dilution to Re values below 0.3 is slow (many hours). The overall process involves a series of three subsequent categories of steps: (i) a rapid (approximately 1-2 min) prevesiculation equilibration of micellar sizes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vesicle to micelle transitions of egg phosphatidylcholine liposomes induced by nonionic surfactants, poly(oxyethylene) cetyl ethers.

Vesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35 mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE(n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.     

Solubilizing effects caused by the nonionic surfactant dodecylmaltoside in phosphatidylcholine liposomes.

The interaction of the nonionic surfactant dodecylmaltoside (DM) with phosphatidylcholine liposomes was investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein released from the interior of vesicles and bilayer solubilization as a decrease in the static light scattered by liposome suspensions. This surfactant showed higher capacity to saturate and solubilize PC liposomes and greater affinity with these structures than those reported for the octyl glucoside. At subsolubilizing level an initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the effective molar ratio of surfactant to phospholipid in bilayers (Re) rose. However, at solubilizing level a direct dependence was established between both parameters. A direct correlation took place in the initial interaction steps (Re up to 0.28) between the growth of vesicles, their fluidity, and Re. A similar direct dependence was established during solubilization (Re range from 0.9 to 1.7) between the decrease in both the surfactant-PC aggregate size, the light scattering of the system, and Re (composition of aggregates). The fact that the free DM concentration at subsolubilizing and solubilizing levels showed values lower than and similar to its critical micelle concentration indicates that permeability alterations and solubilization were determined, respectively, by the action of surfactant monomer and by the formation of mixed micelles.     

Micelle-supported electroenzymology: Succinate dehydrogenation by escherichia coli fumarate reductase in decylubiquinone and octyl glucoside micelles

The concept of micelle-supported electroenzymology is demonstrated using a system consisting of the membrane enzyme Escherichia coli fumarate reductase (FRD), the amphiphilic coenzyme analogue decylubiquinone (DU), the micelle-forming surfactant n-octyl glucoside (OG), and a gold electrode. The OG micelles provide a hydrophobic, membrane mimetic medium for FRD and DU to exchange electrons while the gold electrode serves to regenerate DU. When succinate is presented to the FRD/DU/OG micelle system, electroenzymatic oxidation of succinate to fumarate occurs as evidenced using cyclic voltammetry. DU is shown to be the only electroactive species in the system; and as increasing amounts of succinate are added, the expected increase in the peak anodic (oxidative) current and decrease in the peak cathodic (reductive) current are observed. The peak anodic current approaches a limiting value with succinate concentration in qualitative agreement with simple Michaelis-Menten enzyme kinetics. When the strong competitive inhibitor oxaloacetate is added, enzymatic oxidation of succinate is inhibited as indicated by no change in the peak anodic and cathodic currents with increasing succinate concentration. (c) 1993 John Wiley & Sons, Inc.     

Composition of octyl glucoside-phosphatidylcholine mixed micelles

The composition of mixed micelles of egg phosphatidylcholine (PC) and octyl glucoside was studied by a novel technique based on measuring resonance energy-transfer efficiency between two fluorescent lipid probes present in trace amounts. Equations were derived for calculating the stoichiometry of the composition of mixed micelles from the energy-transfer measurements. These were applied to determining the average number of lipid molecules in the octyl glucoside-egg PC mixed micelle as a function of detergent concentration. The average number of detergent molecules in these mixed micelles was independent of lipid concentration in the range studied (0-500 microM). The dependence of mixed micelle stoichiometry on the concentration of aqueous (monomeric) octyl glucoside is consistent with the assumptions of ideal mixing of the two amphiphiles in the mixed micelles and that mixed micelles can be treated as a distinct phase.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, Re. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the 'equilibrium partition model', the dependence of the 'solubilizing detergent concentration' on the lipid concentration intersects with the lipid axis at -1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical ('solubilizing') effective ratio depends upon the nature of detergent less than any of these two factors.     

Micelle-vesicle transition in phospholipid-bile salt mixtures. A study by precision scanning calorimetry

A systematic study of the micelle-vesicle transformation in phospholipid-bile salt mixtures using differential scanning calorimetry (DSC) indicates that the lipid undergoes a variety of changes in its thermal properties as mixed micellar solutions are diluted to concentrations at which vesicles form. In the experiments, micellar solutions of 50 mg/mL total lipid, containing sodium taurocholate (TC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), are diluted to concentrations corresponding to differing extents of aggregation of the TC with phospholipid. Turbidity and equilibrium dialysis measurements are used to establish boundaries between where micelles persist and where vesicles are formed and to determine the extent of aggregation of the TC with DPPC. At molar ratios Re of bound TC to DPPC greater than 0.3, micellar solutions are formed, while at Re less than 0.15 vesicles are evident upon dilution. As the transformation from micelles to vesicles occurs, the thermal transitions in the lipid change from broad, low Cp (max) peaks in the micelle region to multiple peaks of high cooperativity in regions of composition where lamellar structures and vesicles form. The DSC curves show that in the composition region corresponding to where bilayer micelles exist a new thermal phase forms, which has a melting transition near 32 degrees C, if the solutions are allowed to equilibrate for 48 h at 21 degrees C. Furthermore, at compositions between Re = 0 and 0.25, there is metastability in the lipid when equilibrated at 21 degrees C, but heating the lipid through the thermal transitions leads to reversible behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhodopsin-egg phosphatidylcholine reconstitution by an octyl glucoside dilution procedure

The transmembrane protein bovine rhodopsin was reconstituted with egg phosphatidylcholine (PC) by using a modified detergent dilution technique employing the nonionic detergent octyl-beta-D-glucoside (octyl glucoside). Using this technique, reconstituted membranes having molar phospholipid/protein ratios between 60:1 and 255:1 were prepared. This is in contrast to the results obtained when an octyl glucoside dialysis technique was employed (Jackson, M.L. and Litman, B.J. (1982) Biochemistry 21, 5601-5608). In the latter case, the highest molar phospholipid/protein ratio that could be obtained when reconstituting rhodopsin with egg PC was approximately 50:1. Reconstituted vesicles prepared by the octyl glucoside dilution technique were examined by negative stain and freeze-fracture electron microscopy, and it was found that the vesicles were unilamellar providing the molar PC/protein ratio was below about 200:1, whereas in preparations having ratios higher than this, a significant number of the vesicles were multilamellar. The mean vesicle diameter showed no trend based on the molar PC/protein ratio within the range of 82:1 to 186:1. The mean diameters of the preparations were between 520 and 850 A. Approximately equal numbers of protein particles were observed on the concave and convex fracture faces of the freeze-fracture micrographs of the reconstituted membranes which is indicative of a symmetric distribution of the protein across the bilayer.     

Cholesterol metabolism by purified cytochrome P-450scc is highly stimulated by octyl glucoside and stearic acid exclusively in large unilamellar phospholipid vesicles

Cholesterol side-chain cleavage (CSCC) catalyzed by purified bovine adrenal mitochondrial cytochrome P-450scc is highly dependent on the vesicles that supply cholesterol. Six-fold higher rates are achieved with large unilamellar dioleoylphosphatidylcholine vesicles (diameter 150 nm) prepared by octyl glucoside (OG) dialysis (DOPC-LUV) than with small sonicated vesicles (diameter 30 nm) (DOPC-SUV) (Vmax = 25 and 4 min-1, respectively. Extensive dialysis that may remove OG decreased Vmax rates for DOPC-LUV almost to rates seen with DOPC-SUV. These dialyzed DOPC-LUV were, however, very sensitive to addition of OG (EC50 = 2.5 microM, 4.3-fold stimulation) while DOPC-SUV were only weakly affected (EC50 = 100 microM, 1.6-fold stimulation). This enhancement of CSCC in LUV by OG only occurred when the cholesterol:DOPC exceeded 0.1 and was associated with a 15-fold increase in the Km for cholesterol. Structural changes in both SUV and LUV at high cholesterol:DOPC ratios (0.1-1) were indicated by decreases in internal volume that were insensitive to OG and did not affect the external diameters. Stearic acid produced a similar stimulation of CSCC in LUV (EC50 = 50 microM) and had no effect on SUV. The Vmax for CSCC, produced by OG activation of DOPC-LUV, is comparable to the highest attained for cytochrome P-450scc (Tween 20/cholesterol). In LUV, a minor proportion of OG (1-5% of cholesterol) is thus sufficient to generate a domain of reactive cholesterol that maintains a near-optimum turnover. This increased CSCC was paralleled by increased binding of cholesterol to P-450scc, suggesting that this cholesterol is more readily donated by the membrane to the cytochrome.     

Microviscosity in dilute aqueous solutions of SDS and non-ionic cellulose derivatives of different hydrophobicity: fluorescence probe investigations

The microviscosity in mixed micelles formed in dilute aqueous solutions of sodium dodecyl sulphate (SDS) and a set of non-ionic cellulose ethers of different hydrophobicity has been determined by means of steady-state fluorescence probe techniques. Two hydrophobic probes have been applied in this investigation: 1,3-di(1-pyrenyl)propane (P3P) and perylene. Reference measurements of microviscosity have also been performed on SDS solutions including the uncharged polymers poly(ethyleneoxide) (PEO) or poly(vinylpyrrolidone) (PVP). All compositions investigated showed qualitatively the same general behaviour with an abrupt increase in microviscosity at the critical surfactant concentration where the polymer-surfactant interaction starts (c₁) followed by a maximum and an asymptotically declining region as the surfactant concentration was increased further. Comparison with a recent investigation of a specific ethyl(hydroxyethyl)cellulose (EHEC fraction CST-103)/ SDS/water system (Evertsson & Nilsson (1997) Macromolecules, 30, 2377) revealed that the maximum in microviscosity generally corresponds to a low degree of SDS adsorption (≈ 0.5 mmol of SDS per gram of polymer) and consequently to a high polymer content of the mixed micelles formed in the type of systems studied herein. The hydrophobicity of the cellulose derivatives was found to correlate to the amplitude of the overall microviscosity pattern for the mixed micelles, i.e. an increased polymer hydrophobicity gave an increased rigidity of the polymersurfactant aggregates. An approximately exponential relation was demonstrated between the maxima in microviscosity of the different mixed micelles and the surface activities of the corresponding cellulose derivatives. All polymer/surfactant combinations investigated gave aggregates with a higher rigidity than ordinary SDS micelles. The microviscosity of the mixed micelles of the cellulose derivatives and SDS formed close to c₁ increased as the temperature rose from 20 to 50 °C. This effect was attributed to an increased hydrophobicity of the cellulose ethers upon temperature elevation, hence giving rise to further close-packing of the aggregate structures.     

A biophysical investigation on the binding and controlled DNA release in a cetyltrimethylammonium bromide-sodium octyl sulfate cat-anionic vesicle system

The interactions between cat-anionic (an acronym indicating surfactant aggregates (micelles and vesicles) formed upon mixing cationic and anionic surfactants in nonstoichiometric amounts) vesicles and DNA have been the subject of intensive studies because of their potential applications in biomedicine. Here we report on the interactions between DNA and cetyltrimethylammonium bromide (CTAB)-sodium octyl sulfate (SOS) cat-anionic vesicles. The study was performed by combining dielectric relaxation spectroscopy, circular dichroism, dynamic light scattering, ion conductivity, and molecular biology techniques. DNA is added to positively charged vesicles until complete charge neutralization of the complex and formation of lipoplexes. This occurs when the mole ratio between the phosphate groups of DNA and positive charges on the vesicle is about 1.8. Above this threshold the nucleic acid in excess remains free in solution. This very interesting new result shows that anionic surfactants are not expelled upon saturation, and therefore, no formation of micelles occurs. Furthermore, vesicle-bound DNA can be released in its native form, as confirmed by dielectric spectroscopy and circular dichroism measurements. The nucleic acid is released upon addition of SOS, which competes with the phosphate groups of the DNA: this results in the demolition of the CTAB-SOS cat-anionic vesicles. These results indicate the possibility of a controlled DNA release and might be of interest in biomedicine.     

Vesicles and particles of sodium bis(2-ethylhexyl) phosphate in binary and ternary systems

Vesicles were identified in aqueous solution of pure sodium bis(2-ethylhexyl) phosphate, a short branched chain surfactant. Superficial tension measurements show that the vesicles appear above a molality of 0.02 (0.69 %w). These aggregates are equilibrium structures. The "packing parameter' theory established by Israelachvili et al. allows the prediction of the occurrence of such vesicles. If an organic solvent, such as xylene or ethylhexanoate, is added to the binary system, a different type of aggregate appears, the size of which is determined by several methods including electron microscopy and light scattering. Interfacial tension measurements show that these aggregates would be expected to form above a molality of 0.02. According to our experimental results, the microstructure of these aggregates can be described as micelles and/or vesicles, swollen or not.