STUDIES OF SODIUM TAUROCHOLATE MICELLES USING FLUORESCENT PROBE MOLECULES

Abstract Micelles formed by sodium taurocholate (NaTC) and mixed micelles formed by NaTC with detergents (SDS, reduced Triton X-100 and CTAC) were studied with fluorescent probes. Pyrene was used as an indicator of the polarity of the micellar binding site by comparison of the fluorescence spectra and vibronic band intensity ratios of pyrene in the different systems. Perylene was used as a fluorescence polarization probe to study the rigidity of the NaTC and mixed micelles. The fluorescence lifetime of perylene in the different systems was also measured. Results of the studies were compared with measurements of the probes in cyclohexane, ethanol and aqueous beta-cyclodextrin. Perylene was found to be more rigidly bound in the NaTC micelles than in the detergent micelles. Insertion of small amounts of reduced Triton X-100 into the NaTC micelles appears to increase the rigidity. The binding sites of NaTC and CTAC have similar polarities, and are more polar than those of SDS and reduced Triton X-100. Insertion of any of the detergents into the NaTC micelle decreases the polarity of the binding site, possibly by reducing the penetration of water into the micelle.     

Mass transport in Triton X series nonionic surfactant solutions: a new approach to solute-solvent interactions

Densities, viscosities and tracer diffusion coefficients for solutions of the nonionic surfactants Triton X-45, X-114, X-100 and X-102 in water (except for Triton X-45) and methanol, and for Triton X-100 in three water-methanol mixtures have been measured at 298.15 K and 308.15 K. The activation energy for viscous flow and the contributions to it from solute and solvent have been calculated. Comparison of the Gibbs energies of these systems shows the roles of polyoxyethylene chains of Triton X molecules in the interactions between solute and solvent, and also the effects of solvents on the inter-actions for different solutes. The viscosityB coefficients are positive for all of the surfactant solutions, and the temperature coefficients ofB are negative. In the non-micellar solutions in methanol, values ofB are small and the temperature coefficient ofB is nearly zero. In water-methanol mixtures the critical micelle concentration becomes larger as the methanol content increases up to 40% w/w; micelles are not formed at higher methanol concentrations. TheB coefficient decreases with increasing proportion of methanol in the solvent, and the temperature coefficient ofB changes from a fairly large positive value at low methanol contents to a small negative value at 80% w/w methanol.     

PHOTOREACTIVITY OF ISOLATED PHOTOSYSTEM I PARTICLES UPON COMBINATION WITH ARTIFICIAL LIPID MEMBRANES OR TRITON X-100 MICELLES*

Abstract— PS-I particles isolated according to Shiozawa et al. (1974) show increased rates of O₂-_- and H ⁺-uptake with ascorbate as electron donor upon combination with an artificial vesicular lipid membrane. The amount of increase varies depending on the reconstitution procedure used. Combination of PS-I particles with Triton X-100 micelles increases these photochemical activities even more. The observed proton uptake in PS-I lipid vesicles is not caused by the well-known proton gradient found in thylakoid membranes, since lipid vesicles containing extracted leaf Chl show the same activities and uncouplers have no effect. Because these phenomena are also caused by solubilized Chl, it is concluded that there is no obvious correlation with PS-I activity. Proton uptake most probably is caused by oxidation of ascorbate by either singlet oxygen, superoxide or OH-radicals formed in the light. Experimental results are obtained which indicate that Chl in lipid catalyzes formation of superoxide and singlet oxygen. However, it is not clear whether superoxide formation is caused by direct electron transport from excited Chl to oxygen or by a secondary reaction. Diphenylcarbazone disproportionation has been reported as a specific photosystem I reaction. However, PS-I lipid vesicles and Chl-lipid-Triton X-100 mixtures oxidize DPCN at comparable rates, showing that the reaction is not specific for PS-I. Cations stimulate DPCN disproportionation in Chl-lipid-Triton X-100 mixtures but do not affect the rate of P700 photooxidation at all. Therefore it is suggested that Gross and Greniers (1978) conclusion that cation regulation of PSI electron flow (studied by DPCN disproportionation of PS-I particles in Triton X-100 micelles) provides a fine tuning mechanism for energy transfer, has to be reevaluated.     

Action of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis is significantly influenced by coexisting lipids in substrate-detergent micelles.

The effects of phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol on the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail in phosphatidylinositol (PI)/detergent mixed micelles. By addition of PC, the enzymatic hydrolysis of PI was significantly stimulated in PI/Triton X-100 as well as PI/sodium deoxycholate (SDC) mixed micelles. SM stimulated enzyme activity toward PI/Triton X-100 micelles at a lower molar ratio of SM to PI, but was rather inhibitory at a ratio higher than 2.0. The enzyme activity became significantly lower with an increase of PE or cholesterol in PI/Triton X-100 micelles. Actually, both PE and cholesterol were intensively inhibitory when added at a higher molar ratio to PI in Triton X-100-containing micelles. In the system of PI/SDC mixed micelles, not only PC but also SM, PE and cholesterol enhanced the enzymatic hydrolysis of PI. The difference between PI/Triton X-100 and PI/SDC micelles regarding the effects of these lipids on PI-PLC action, must be dependent on the physical state of micelles formed by these detergents and lipids.     

Formation of Copper(II) Thiocyanato and Cadmium(II) Iodo Complexes in Micelles of Nonionic Surfactants with Varying Poly(ethylene oxide) Chain Lengths

Formation of copper(II) thiocyanato and cadminum(II) iodo complexes in micelles of poly(ethylene oxide) (PEO)-type nonionic surfactants with varying PEO chain lengths of 9.5 (Triton X-100), 30 (Triton X-305), and 40 (Triton X-405) has been studied by titration spectrophotometry and calorimetry at 298 K. In a given surfactant solution, all data obtained were analyzed by assuming formation of ternary complexes MX(n)Y(m)((2-n)+) (M = Cu(II),Cd(II); X = SCN(-), I(-); Y = surfactant), and the complexes thus form in aqueous phase (m = 0) or in micelles (m = 1). In the Cu(II)-SCN(-) system, spectrophotometric data obtained by varying concentrations of the surfactant can be explained well in terms of formation of Cu(NCS)(2)Y in micelles and Cu(NCS)(+) and Cu(NCS)(2) in an aqueous phase, and it turned out that formation constant of Cu(NCS)(2)Y increases with increasing PEO chain length. In the Cd(II)-I(-) system, the formation of CdI(3)Y(-) and CdI(4)Y(2-) is concluded in micelles, and that of CdI(+), CdI(3)(-), and CdI(4)(2-) in an aqueous phase. Interestingly, formation enthalpies of CdI(3)Y(-) and CdI(4)Y(2-) become significantly less negative with increasing PEO chain length. This suggests that transfer of the complexes from aqueous solution to a hydrophobic octylphenyl (OP) moiety in micelles is significantly more exothermic than that to a hydrophilic PEO one. Thermodynamic parameters of transfer of CdI(3)(-) and CdI(4)(2-) from aqueous solution to the OP and PEO moieties of micelles have been evaluated. Copyright 2000 Academic Press.     

Spectral and electrochemical studies of bis(diimine)copper(II) complexes in anionic, cationic and nonionic micelles

The spectral and redox behavior of bis(diimine)copper(II) complexes, where diimine is bipyridine, 1,10-phenanthroline, 4-methyl-1,10-phenanthroline, 5-methyl-1,10-phenanthroline, 5-nitro-1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline, 5,6-dimethyl-1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline, 3,4,7,8-tetramethyl-1,10-phenanthroline and dipyrido-[3,2-d:2',3'-f]-quinoxaline, are significantly different in aqueous and in aqueous SDS, CTAB and Triton X-100 micellar solutions. The (1)H NMR spectral study in aqueous (D(2)O) and aqueous micelles reveals that the Cu(II) complexes interact more strongly with SDS than with CTAB and Triton X-100 micelles and at sites on SDS micelles different from those on the latter. Ligand Field spectral studies reveal that the complexes exist as the dicationic aquated species [Cu(diimine)(2)(H(2)O)(2)](2+), which interacts strongly with the anionic SDS micelles through columbic forces. However, they exist as [Cu(diimine)(2)(H(2)O)Cl](+) and/or [Cu(diimine)(2)H(2)] located in the hydrophobic microenvironments in Triton X-100 and CTAB micelles. The attainment of reversibility of the redox systems in the micellar microenvironments is remarkable and this illustrates that the Cu(II) and Cu(I) species undergo stereochemical changes suitable for reversible electron-transfer. The remarkable differences in spectral and electrochemical properties of Cu(II) complexes in aqueous and aqueous micellar solutions illustrate that the complexes are nestled largely within the micellar environments and imply that the accessibilities of the complexes to electron-transfer are different and are dependent on the nature of micelles as well as the nature and hydrophobicity of the diimine ligands.     

三磺化酞菁在微乳液、胶束、醇-水体系中的二聚现象研究

本文采用分光光度法研究了三磺化酞菁(S3Pc)在微乳液(Triton X-100-壬烷-正戊醇-水)、胶束(Triton X-100)、醇(甲、乙、丙醇)-水体系中的二聚现象。计算了S3Pc的二聚常数Kd。结果表明, S3Pc的Kd随着微乳液、胶束中表面活性剂的浓度的增加而减小; 随着醇-水溶液的羟基摩尔浓度(Mo-h)的增加而增加。     

Comprehensive examination of mesophases formed by DMPC and DHPC mixtures

Mixtures of long- and short-chain phospholipids, specifically 14:0 and 6:0 phosphatidylcholines (DMPC and DHPC), have been used successfully in NMR studies as magnetically alignable substrates for membrane-associated proteins. However, recent publications have shown that the phase behavior of these mixtures is much more complex than originally thought. Using polarized light microscopy and small-angle neutron scattering, phase diagrams of DMPC/DHPC mixtures at molar ratios of 2, 3.2, and 5 have been determined. Generally, at temperatures below the main-chain melting transition of DMPC (T(M) = 23 degrees C), an isotropic phase of disk-like micelles is found. At high temperatures (T > 50 degrees C), a lamellar phase consisting of either multilamellar vesicles (MLV) or extended lamellae is formed, which at low lipid concentrations (e.g., MLV) coexists with an excess of water. At intermediate temperatures and lipid concentrations, a chiral nematic phase made up of worm-like micelles was observed.     

Self-aggregation and supramolecular structure investigations of Triton X-100 and SDP2S by NOESY and diffusion ordered NMR spectroscopy

The self-aggregation and supramolecular micellar structure of two surfactants in aqueous solution, the anionic surfactant SDP2S (sodium dodecyl dioxyethylene-2 sulfate) and the nonionic surfactant Triton X-100 (octylphenol-polyoxyethylene ether with 9.5 ethoxy groups), were investigated by NMR spectroscopy. The critical micellar concentration (CMC), the size, and shape of the aggregates were determined by diffusion ordered NMR spectroscopy (DOSY), while 2D NOESY NMR spectra were used to study the mutual spatial arrangement of surfactant molecules in the aggregated state. A nonlinear increase of the micellar hydrodynamic radius, indicating possible sphere-to-rod shape transition, was found for SDP2S at higher surfactant concentrations. Triton X-100 micelles were found to be almost spherical at low surfactant concentrations, but formation of ellipsoid shaped particles and/or micellar aggregation was observed at higher concentrations. The NOESY data show that at low concentration Triton X-100 forms a two-layer spherical structure in the micelles, with partially overlapping internal and external layers of Triton X-100 molecules and no distinct hydrophilic-hydrophobic boundary.     

Assembly properties of Triton X-100/phosphatidylcholine aggregates during liposome solubilization

The assembly properties of the nonionic surfactant Triton X-100 and phosphatidylcholine (PC) aggregates during the overall solubilization process of PC liposome were investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein (CF) released from the interior of vesicles and bilayer solubilization as a decrease in the static light scattered by liposome suspensions. A direct dependence was established between the bilayer/aqueous phase surfactant partition coefficients (K), the growth of vesicles and the leakage of entrapped CF in the initial interaction steps (surfactant to phospholipid molar ratioRe up to 0.2). These changes may be related to the increasing presence of surfactant molecules in the outer monolayer of vesicles. In theRe range 0.2–0.35 the coexistence of a low vesicle growth with a constant increase of CF release may be correlated with the decrease inK (increased rate of flip-flop of surfactant molecules). Furthermore, in theRe range between 0.64 and 2.0 (lytic levels) almost a linear dependence was detected between the composition of these aggregates (Re) and the decrease in both the surfactant-PC aggregate size and the static light scattered by the system. This dependence was not observed in the last solubilization steps (Re range 2.0–2.60) possibly due to the increased formation of mixed micelles in this interval. The fact that the free Triton X-100 concentration at sublytic and lytic levels showed respectively lower and similar values than its critical micelle concentration confirms that permeability alterations and solubilization were determined respectively by the action of surfactant monomer and by the formation of mixed micelles.Abbreviations PC phosphatidylcholine - PIPES piperazine-1,4 bis(2-ethanesulphonic acid) - T_X-100 Triton X-100 - CF 5(6)-carboxyflucrescein - Re enective surfactant/lipid molar ratio - Re _SAT effective surfactant/lipid molar ratio for bilayer saturation - Re _SOL enective surfactant/lipid molar ratio for bilayer solubilization - S _W surfanctant concentration in the aqueous medium - S _B surfactant concentration in the bilayers - S _T total surfactant concentration - K bilayer/aqueous phase surfactant partition coefficient - K _SAT bilayer/aqneous phase surfactant partition coefficient for bilayer saturation - K _SOL bilayer/aqueous phase surfactant partition coefficient for bilayer solubilization - PL phospholipid - TLC-FID thinlayer chromatography/flame ionization detection system - PI polydispersity index - CMC critical micellar concentration - r ² regression coefficient     

Ion-exchange chromatography separation of the detergent and the solvent from immunoglobulins after solvent-detergent treatment.

For inactivation of lipid-enveloped viruses during the immunoglobulin production, the solvent-detergent (S/D) method was applied. Tri-n-butyl phosphate (solvent) and Triton X-100 (detergent) were removed from S/D treated immunoglobulins by ion-exchange chromatography on Q-Sepharose Fast Flow (FF). During the chromatographic procedure immunoglobulins remained bound on a Q-Sepharose FF, whereas solvent and detergent were eluted by washing with starting buffer. Elution of immunoglobulins was achieved by increasing the ionic strength of the starting buffer. The final immunoglobulin preparation contained less than 10 microg/ml of Triton X-100 and less than 2 microg/ml tri-n-butyl phosphate. It was confirmed that the S/D procedure did not cause a significant change in polymers and specific antibodies content. Immunoglobulin classes were also not affected by the same procedure.     

Micelle‐Triggered β‐Hairpin to α‐Helix Transition in a 14‐Residue Peptide from a Choline‐Binding Repeat of the Pneumococcal Autolysin LytA

Choline‐binding modules (CBMs) have a ββ‐solenoid structure composed of choline‐binding repeats (CBR), which consist of a β‐hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline‐binding ability, we have analysed the third β‐hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native‐like β‐hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α‐helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β‐hairpin to α‐helix conversion is reversible. Given that the β‐hairpin and α‐helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this “chameleonic” behaviour is the only described case of a micelle‐induced structural transition between two ordered peptide structures.     

Micelles,Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.     

Self-organization of amide dendrons and their dendronized macromolecules

A polymerizable methacryl unit was introduced at the focal moiety of the amide dendrons which have amide branches and alkyl periphery. Their dendronized polymers were also prepared by the radical polymerization of the methacryl units. The self-organization characteristics of dendrons and dendronized polymers were then investigated in both the organic and aqueous phases. The amide dendrons (1M and 2M) in which the focal carboxyl group was blocked with methacryl units did not form gel in organic media such as chloroform or THF, whereas amide dendrons with a free carboxyl group at the focal point form self-organized structures. In the aqueous phase, 1M and 2M formed spherical vesicular assemblies. The dendronized polymers with first and second generation dendrons, 1P and 2P, respectively, exhibited lamellar and columnar organization in toluene. In addition to hydrogen bonding between the dendritic amide branches and van der Waals interactions between the alkyl periphery, steric confinement of dendritic side groups along the polymer backbone played a key role in the packing process of the dendronized polymers. In aqueous phase, 1P and 2P showed spherical vesicular aggregates with persistent stability in the presence of Triton X-100.     

Transfer of coenzyme Q0 from water to aqueous surfactant solutions: the case of Triton X-100

The effect of UQ0 on the micellization equilibrium of Triton X-100 has been studied by the analysis of the UV absorption spectra of Triton X-100. In the range of the UQ0 concentration investigated, the critical micelle concentration (CMC) increases at increasing of the solute concentration. The dependence of the CMC on UQ0 concentration has been used to calculate the generalized Setchenov constant. Mixing and dilution enthalpies of aqueous solutions of UQ0 and Triton X-100 were measured and used to calculate the enthalpies of transfer of UQ0 from water to Triton X-100 aqueous solutions. From the dependence of the enthalpy of transfer on surfactant concentration, the distribution constant between aqueous and micellar phase and the standard enthalpy of transfer from water to Triton X-100 micelles were evaluated along with the standard transfer free energy and entropy. All measurements were carried out at 298 K.     

Dimensions and Orientation of Triton X-100 Micelles in Aqueous Solutions,According to Turbidimetric Data

Russian Journal of Physical Chemistry A - The size and orientation of micelles of nonionic surfactant Triton X-100 in aqueous solutions is determined turbidimetrically. It is shown that micelles...     

Proteomics of rat liver Golgi complex: minor proteins are identified through sequential fractionation

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.     

Phase dependent electrochemical properties of polar self-assembled monolayers (SAMs) modified via the fusion of mixed phospholipid vesicles

Unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and varying quantities of either 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol) (sodium salt) (DMPG) or 1,2-dimyristoyl-3-trimethylammonium-propane (chloride salt) (DMTAP) were used to deposit lipid bilayer assemblies on self-assembled monolayers (SAMs) on gold. The supporting SAMs in turn were composed of ferrocene-functionalized hexadecanethiol chains (FcC16SH) diluted to low coverage in 1-hydroxylhexadecanethiol (HOC16SH) or a single-component monolayer phase of the latter. The mass coverages of the DMPC/DMPG layers deposited in this way were measured using surface plasmon resonance (SPR) and found to decrease with an increasing content of DMPG in the vesicles. The SPR data show that the lipid assembly, while stable with respect to gentle rinsing in aqueous buffer, is reversible and the lipid adlayer is removable by immersion in a solvent such as ethanol. The effects of the adsorbed lipid layer on the electrochemical interactions of the hybrid lipid/SAM with several redox probes [e.g., K4Fe(CN)6, Ru(NH3)6Cl3, and CsHsFe-[(C5H4CH2N+H(CH3)2] were characterized using cyclic voltammetry (CV). At a composition of 5% DMPG in DMPC, the permeabilities of the probes through the lipid layer were affected significantly relative to that observed with a pure DMPC layer. These effects include a striking observation of an enhanced, ionic-charge-specific molecular discrimination of the electrochemical probes. At higher concentrations of the DMPG, significant permeation of the lipid adlayer was seen for all the probes. These latter changes are also attended by a significant increase in the capacitive currents measured in CV experiments as compared to those observed for either a pure SAM or one modified by only DMPC. This effect likely results from the influence of the charged lipid on the diffuse Gouy-Chapman electrolyte layer at the SAM interface. In contrast to the behaviors seen with DMPG, the incorporation of DMTAP into the adsorbed DMPC had no impact on the permeation of the adlayer by soluble redox probes as judged by the observed electrochemistry, a result that appears to correlate with a less ideal mixing of lipids in the DMPC/DMTAP system relative to that of a DMPC/DMPG mixture.     

Do electrospray mass spectra of surfactants mirror their aggregation state in solution?

One important feature in the gas phase chemistry of surfactants is to ascertain whether their aggregates produced by electrospray ionization reflect those formed in the starting solution. With this aim, we have performed ESI-MS, ESI-MS/MS and ER-MS spectra of bis(2-ethylhexyl)sulfosuccinate (AOTNa) solutions in different solvents, i.e. water, water/methanol, methanol and n-hexane. The results clearly indicate that, notwithstanding the strongly different aggregation state in solution (direct micelles in water and in water/methanol, molecular dispersion in methanol and reverse micelles in n-hexane) and marked effects of the solvent polarity on the total ionic current, the surfactant aggregates in gas phase show identical structural features. Analogous conclusions can be drawn analyzing the infrared multiple photon dissociation (IRMPD) spectra of AOTNa solutions in water/methanol and n-hexane. Moreover, according to the idea that gas phase can be considered an apolar environment par excellence, data consistently suggest a reverse micelle-like aggregation. Some peculiarities of the mechanisms leading to aggregate formation through electrospray ionization of surfactant solutions in solvent media with different polarity have been also discussed.     

Carotenoid radical anions and their protonated derivatives

In this study, we report the protonation reactions for astaxanthin and canthaxanthin radical anions in methanol, alkaline methanol, and aqueous 2% Triton X-100 at different pH values. The pKa values for the corresponding alpha-hydroxy radical derivatives of astaxanthin, canthaxanthin, and beta-apo-8'-carotenal were estimated in 2% Triton X-100. Also, the effects of the microenvironment and the structure of the carotenoids on the protonation rate constant are discussed.