Onset Of Solubilization Research Articles

Lipid Scrambling Induced by Membrane-Active Substances

The functional roles of the lipid asymmetry of biomembranes are attracting increasing attention. This study characterizes the activity of surfactants to induce transmembrane flip-flop of lipids and thus “scramble” this asymmetry. Detergent-induced lipid scrambling of liposomes mimicking the charge asymmetry of bacterial membranes with 20 mol % of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol in the outer leaflet only was quantified by ζ-potential measurements for octaethylene glycol dodecyl ether (C12EO8), octyl glucoside (OG), and dodecyl maltoside. Membrane leakage was separately measured by the fluorescence lifetime-based calcein leakage assay and the onset of the membrane-to-micelle transition by isothermal titration calorimetry. Partition coefficients and partial molar areas were obtained as well. For the quickly membrane-permeant C12EO8 and OG, leakage proceeds at a rather sharp threshold content in the membrane, which is well below the onset of solubilization and little dependent on incubation time; it is accompanied by fast lipid scrambling. However, unlike leakage, flip-flop is a relaxation process that speeds up gradually from taking weeks in the detergent-free membrane to minutes or less in the leaking membrane. Hence, after 24 h of incubation, 10 mol % of C12EO8 or 50 mol % of OG in the membrane suffice for virtually complete lipid scrambling, whereas leakage remains below 10% for up to 14 mol % of C12EO8 and 88 mol % of OG. There is thus a concentration window in which lipid scrambling proceeds without leakage. This implies that lipid scrambling must be considered a possible mode of action of antimicrobial peptides and other membrane-active drugs or biomolecules. A related, detergent-based protocol for scrambling the lipid asymmetry of liposomes and maybe cells without compromising their overall integrity would be a very valuable tool to study functions of lipid asymmetry.

Octyl-β-D-Glucopyranoside Shows Composition Dependent Disordering Effects in Ternary Lipid Bilayers

Solubilization of lipid bilayers by the nonionic detergent OG has been well characterized in one and two component lipid systems, but has not been extensively studied in ternary membranes containing mixed fluid phases. In order to understand the effects of membrane order on solubilization by OG, LUVs of pure POPC were compared to those composed of POPC plus varying fractions of sphingomyelin (SM) and cholesterol. Detergent partitioning from the aqueous phase into the bilayer and membrane solubilization were monitored by ITC. The fluorescent membrane probe, DPH, was observed by time-resolved fluorescence intensity and polarization decay in the presence of increasing detergent concentrations to determine the effects on membrane order and dynamics. The results of partitioning experiments showed approximately a 50% decrease in the partition constant between pure POPC and the most ordered samples. ITC solubilization experiments showed clear boundaries for the micelle-bilayer mixed aggregate coexistence region in samples of low initial order, but the distinct thermodynamic signature associated with this coexistence region was not present in the cholesterol and SM rich samples. The average excited state lifetime of DPH, an indicator of water penetration, in vesicles rich in POPC showed a rapid increase at the onset of solubilization, whereas vesicles rich in SM and cholesterol showed a corresponding rapid decrease. Dynamic fluorescence depolarization data analyzed in terms of a Brownian rotational diffusion model revealed an increase in the occupancy of the bilayer midplane by DPH prior to solubilization in less ordered membranes coupled with a constant rotational correlation time. Membranes with higher initial order showed little increase in midplane occupancy and increasing rotational correlation time in the same detergent concentration range, suggesting that two modes of membrane disruption are possible and depend on the lipid makeup of the bilayer.

Superior Membrane Permeabilization and Solubilization Properties of Biosurfactants may be Explained by Heterogeneous Perturbation

Biosurfactants such as antimicrobial lipopeptides, saponins, and bile salts often show a superior performance to permeabilize and lyse membranes and/or a better suitability for membrane protein solubilization than classical, synthetic detergents. We propose a classification of surfactants into those that are homogeneously disordering versus heterogeneously perturbing a given membrane to help explaining and predicting this behaviour. Typical synthetic detergents such as C12EO8, octyl glucoside, SDS, and lauryl maltoside were identified as homogeneously disordering by the limiting fluorescence anisotropy of several DPH derivatives reaching a characteristic, low level at the onset of solubilization. The biosurfactants surfactin, fengycin, iturin, digitonin, and lysophosphatidylcholine along with the synthetic CHAPS belong to another class that initiates membrane lysis without critical disordering the whole membrane. They disrupt the membrane locally due to a spontaneous segregation from the lipid and/or an ordering effect that induces packing defects. This may account for enhanced activity, selectivity, and mutual synergism of antimicrobial biosurfactants. They should also be prone to form partially-demixed, asymmetric micelles (or bicelles) with a relatively lipid- (and sterol?) rich core surrounding a solubilized protein. Triton shows the pattern of a segregating surfactant in the presence of cholesterol. Zeta potential measurements of liposomes exposed to surfactin show strong peptide-peptide interactions already at a few mol-% in the membrane.

Membrane properties of plant sterols in phospholipid bilayers as determined by differential scanning calorimetry, resonance energy transfer and detergent-induced solubilization

The increased use of plant sterols as cholesterol-lowering agents warrants further research on the possible effects of plant sterols in membranes. In this study, the effects of the incorporation of cholesterol, campesterol, β-sitosterol and stigmasterol in phospholipid bilayers were investigated by differential scanning calorimetry (DSC), resonance energy transfer (RET) between trans parinaric acid (tPA) and 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl- sn-glycero-3-phosphocholine (NBD-PC), and Triton X-100-induced solubilization. The phospholipids used were 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), d- erythro- N-palmitoyl-sphingomyelin (PSM), and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC). In DSC experiments, it was demonstrated that the sterols differed in their effect on the melting temperatures of both the sterol-poor and the sterol-rich domains in DPPC and PSM bilayers. The plant sterols gave rise to lower temperatures of both transitions, when compared with cholesterol. The plant sterols also resulted in lower transition temperatures, in comparison with cholesterol, when sterol-containing DPPC and PSM bilayers were investigated by RET. In the detergent solubilization experiments, the total molar ratio between Triton X-100 and POPC at the onset of solubilization ( R t,sat) was higher for bilayers containing plant sterols, in comparison with membranes containing cholesterol. Taken together, the observations presented in this study indicate that campesterol, β-sitosterol and stigmasterol interacted less favorably than cholesterol with the phospholipids, leading to measurable differences in their domain properties.

Comparison of Triton X-100 Penetration into Phosphatidylcholine and Sphingomyelin Mono- and Bilayers

This study has addressed the question whether there is a difference in resistance to Triton X-100 penetration of sphingomyelin and phosphatidylcholine membranes. We have studied this by measuring the penetration of Triton X-100 into monolayers of either d-erythro-N-palmitoylsphingomyelin or dipalmitoylphosphatidylcholine (DPPC) at constant lateral surface pressure and 22 °C. In addition, we also compared how Triton X-100 penetrated into egg phosphatidylcholine and d-erythro-24:1Δ15c sphingomyelin monolayers and how the presence of cholesterol affected the penetration process. In bilayer membranes we assessed Triton X-100 insertion and solubilization by studying the thermodynamics of the partition process and determining the detergent:ratio at which the solubilization is initialized. The penetration of Triton X-100 from the subphase into the monolayers was concentration and surface pressure dependent. The penetration increased linearly with time and concentration for DPPC monolayers, but with sphingomyelin monolayers, the response was clearly biphasic and faster. If the penetration was allowed to proceed, the monolayers eventually became unstable and collapsed (at collapse the detergent-to-lipid ratio was about 1:4 in the monolayer). Inclusion of 30 mol % cholesterol in the monolayers made them more resistant to Triton X-100 penetration, irrespective of the host phospholipid type. In bilayers the partition coefficient (K) becomes lower and the enthalpy change (ΔH) higher for partitioning of Triton X-100 into d-erythro-N-palmitoylsphingomyelin as compared to into DPPC. Onset of solubilization was reached at lower detergent:lipid ratios in d-erythro-N-palmitoylsphingomyelin than in DPPC vesicles. In conclusion, we have observed that d-erythro-N-palmitoylsphingomyelin monolayers do not show increased resistance toward Triton X-100 penetration as compared with membranes prepared from an acyl-matched glycerophospholipid, at least not at room temperature and not even when cholesterol is present. Sphingomyelin bilayers are more susceptible to Triton X-100 solubilization than phosphatidylcholine bilayers, although K was lower for Triton X-100 into sphingomyelin bilayers.

Detergent-mediated reconstitution of membrane proteins.

The efficiency of reconstitution of the lactose transport protein (LacS) of Streptococcus thermophilus is markedly higher with Triton X-100 than with other detergents commonly employed to mediate the membrane insertion. To rationalize these differences, the lipid/detergent structures that are formed in the reconstitution process were studied by cryotransmission electron microscopy. Surprisingly, the two nonionic detergents Triton X-100 and n-dodecyl beta-D-maltoside (DDM) affected the liposome structures in a completely different manner. Preformed liposomes titrated with Triton X-100 maintained their bilayer structure far beyond the onset of solubilization, and transport activity was maximal when LacS was inserted into these structures. With DDM the vesicular structures were already disrupted at the onset of solubilization and these membrane sheets were converted into long threadlike micelles at higher DDM to lipid ratios. Triton X-100 allowed the protein to be reconstituted with the hydrophilic surface exposed to the outside, whereas LacS was incorporated randomly when DDM was used. These differences in orientation are readily explained by the different lipid-detergent structures formed by Triton X-100 and DDM. The orientation of the reconstituted LacS protein is a critical factor for the activity of the protein as the kinetics of translocation is very different for opposite directions of transport.

Effective detergent/ chlorophyll ratio and detergent concentration in the aqueous phase during solubilization of Phormidium laminosum membranes

Experiments of turbidity decrease induced by detergents were systematically performed to characterize the solubilization of Phormidium laminosum membrane fragments. SDS, Triton X-100 and a mixture of octyl glucoside/decyl maltoside/lithium dodecyl sulfate (OG/DM/LiDS, in a molar ratio of 4.19:2.54:1) were used. The detergent concentration in the aqueous phase ( D w) and the effective detergent/chlorophyll ratio in mixed aggregates (R e) were determined. Both parameters increased during the solubilization and in an exponential way in the range from 10 to 90% solubilization. At detergent concentrations which caused the complete solubilization, D W values were close to the described critical micellar concentrations (cmc), but solubilization started at concentrations well below the cmc. At the onset of solubilization five molecules of SDS, one of Triton X-100 and three of the mixture OG/DM/LiDS, per chlorophyll molecule, saturated the membrane fragments. The increase of D W and R e values was characterized by two constants. This permits the design of a model to predict the detergent concentration which produces a desired solubilization of thylakoid membrane fragments for a given chlorophyll concentration.

Binding of a nonionic detergent to membranes: flip-flop rate and location on the bilayer.

The kinetic aspects of amphiphile interaction with intact membranes (unilamellar and multilamellar liposomes, sarcoplasmic reticulum vesicles) were studied, with the nonionic detergent octa(ethylene glycol) dodecyl monoether (C12E8) as a prototype. C12E8 was bound to these membranes noncooperatively and with a maximum of 0.6-0.8 mol per mole of phospholipid, before the onset of solubilization. Binding was not affected by ultrasonication to expose internal binding sites on the inner leaflet. All detergent could be removed from the membranes by treatment with hydrophobic beads. Furthermore, bound detergent, also from the inside of multilayered liposomes, comprising 10-20 bilayers, was quickly released by dilution of the membranes, followed by gel filtration. The time course of these processes was investigated with a rapid-filtration apparatus, using glass fiber filters to deposit membrane material. Both detergent binding and removal could be described by a monoexponential process with a half-time of approximately 350 ms for all types of membranes. Binding of detergent enhanced the intrinsic fluorescence of sarcoplasmic reticulum vesicles. This occurred in less than 100 ms, probably as the result of direct interaction of C12E8 with Ca2+-ATPase at a few binding sites. The data show that flip-flop of C12E8 across lipid membranes is a rapid process that cannot account for incomplete detergent removal in reconstitution experiments [Ueno, M., Tanford, C., & Reynolds, J. A. (1984) Biochemistry 23, 3070-3076]. It is also suggested that other nonionized amphiphiles, including those with an anesthetic action, rapidly gain access to membrane proteins on the inside of the cell, even when used at low, clinical doses.