HYDROPHOBIZING EFFECT OF CATIONS ON ACIDIC PHOSPHOLIPID MEMBRANES

By means of contact angle measurements with water and aqueous salt solutions, it is shown that plurivalent cations increase the hydrophobicity of negatively charged phospholipid vesicle membranes (consisting of phosphatidic acid, PA, or of phosphatidylserine, PS), but does not influence the hydrophobicity of neutral phospholipid membranes, (e.g., phosphatidylcholine, PC, at up to 200 mM of CaCl₂). The hydrophobizing action of cations on PA and PS membranes is concomitant with the reduction in (negative) zeta potential with increasing cation concentrations. Trivalent cations, La³⁺, showed more effective in hydrophobizing negatively charged phospholipid membranes than divalent and monovalent cations. Except for hydrogen ions, monovalent cations do not show any appreciable hydrophobizing effect on lipid vesicle membranes at concentrations less than 1 M. The hydrophobizing effect on phospholipid membranes can also be used to explain the induction of lateral phase separation into patches of different phospholipids as well as cell fusion.     

Differences in permeability of dioleoylphosphatidylcholine model membranes containing saturated or unsaturated phosphatidic acids

At low millimolar Ca²⁺ concentrations, large unilamellar dioleoylphosphatidylcholine vesicles containing dimyristoylphosphatidate (20 mol%) release enclosed solutes like sulphate, but influx of Ca²⁺ is not demonstrable. Coincident with the permeability change, vesicle aggregation and membrane fusion are observed. These results contrast with those for dioleoylphosphatidate-containing vesicles under the same conditions, which show Ca²⁺ influx and Ca²⁺ chelator efflux, but no sulphate efflux, vesicle aggregation or membrane fusion. The observed differences in permeability behaviour of membranes containing these two phosphatidate molecular species are discussed with respect to the differences in their phase behaviour.     

Activity coefficients of CaCl2 and MgCl2 in the presence of dipalmitoylphosphatidylcholine-phosphatidylinositol vesicles in aqueous media

Mean activity coefficients of CaCl₂ and MgCl₂ salts have been measured in the presence of phospholipid vesicles, by use of the EMF method with ion-exchange membrane electrodes, as a novel procedure for studying the direct interactions between electrolytes and phospholipid vesicles. Mixed lipid sonicated vesicles have been prepared from dipalmitoylphosphatidylcholine (DPPC)-phosphatidylinositol (PI) mixtures, covering a range of composition. The CaCl₂ and MgCl₂ concentration range studied was 0.01−0.001 mol l⁻¹. For both salts studied, the activity coefficients decrease linearly as the vesicle concentration in the suspension increases while the concentration of salt was kept constant. Maintaining a constant vesicle concentration, the decrease in the activity coefficients is more pronounced the more dilute the concentration of the salts. Quantitative differences were observed in the behaviour of both salts which are explained on the basis of a higher affinity of the Ca²⁺, compared with that of Mg²⁺ ions, to the vesicle surface. Using a simple model for the vesicle-salt systems, the number of cations specifically bound to the vesicles has been calculated. In view of the results it can be concluded that estimation of the activity coefficients of the salts is a useful method for the study of the interactions taking place in vesicle-salt systems.     

Interaction and aggregation of lipid vesicles (DLVO theory versus modified DLVO theory)

Interaction and aggregation of acidic phospholipid (phosphatidylserine) vesicles were studied with variation of cation species and their concentrations in vesicle suspensions, and of vesicle sizes. Aggregation was determined by measuring turbidity of vesicle suspension. The experimental results of aggregation of vesicles induced by monovalent cations (Na⁺, K⁺, Cs⁺ and TMA⁺) were explained well in terms of the interaction energy of two interacting vesicles using the ordinary Derjaguin–Landau–Verwey–Overbeek (DLVO) theory for both small and large lipid vesicles. However, the experimental results of aggregation of vesicles induced by divalent cations (Ca²⁺, Mg²⁺ and Ba²⁺) were not explained by the ordinary DLVO theory. In order to explain the experimental results of these vesicle aggregation phenomena, it was necessary to modify the theory by including hydration interaction energies which are due to hydrated water at membrane surfaces, and their magnitude and sign depend upon the nature (hydrophobicity) of the membrane surface.     

Cation-mediated interaction of dextran sulfate with phospholipid vesicles: binding, vesicle surface polarity, leakage and fusion

 The interaction of dextran sulfate (DS) with dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles was investigated. DS of different molecular weights (1, 8, 40 and 500 kDa) and divalent cations (Ca²⁺, Mg²⁺ and Mn²⁺) and the trivalent cation La³⁺ were used in the experiments. Binding of DS was studied by use of the microelectrophoresis and monolayer technique. Binding depends strongly on cation and NaCl concentrations in the medium and does not occur in the absence of multivalent cations. Binding is modulated by the molecular weight of the polymers; DS with lower molecular weights lead to less negative zeta potentials at identical concentrations. A comparable monomer of DS, glucose-6-sulfate, does not change the zeta potential of DMPC vesicles. Monolayer experiments revealed a decrease in surface pressure after addition of multivalent cations and DS, indicating a stronger interaction of the cation–polymer complex with the phosphatidylcholine headgroups than its penetration into the phospholipid (PL) bilayer. The cation-mediated binding of DS to the vesicles leads to aggregation of the vesicles. The tendency to promote aggregation of DMPC vesicles is La³⁺>Ca²⁺>Mn²⁺≥ Mg²⁺. The aggregated vesicles show a stacklike arrangement of the bilayers as shown by freeze-fracture electron microscopy. The strong aggregation is accompanied by lipid mixing measured by the 1,4-nitrobenzo-2-oxa-1,3-diazole–phosphatidylethanolamine (PE)/lissamine rhodamine B sulfonyl-PE assay. At low ionic strength substantial lipid mixing can be observed in the previously mentioned order of the cations. This lipid mixing is accompanied by an increase in the permeability of the vesicles as revealed by the 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylenebis (pyridiium bromide) assay. The extent of leakage is determined by the cation used and the DS molecular weight. These interaction processes between the opposing bilayers are connected with a decrease in the water content in the gap between the opposing PL bilayers. As a measure for the change of the polar properties of the vesicle surface the shift of the emission wavelength of the fluorescent probe dansylphosphatidylethanolamine was measured. The effectiveness of divalent/trivalent cations to decrease the surface dielectric constant of DMPC vesicles also followed the sequence of ions as found for binding, PL mixing and leakage. The results are discussed in terms of the changed hydration at the bilayer surface induced by DS in the presence of multivalent ions. Received: 16 December 1998/Accepted: 17 December 1999     

Phospholipase D-mediated aggregation, fusion, and precipitation of phospholipid vesicles

Large unilamellar vesicles with a diameter of 100 nm were prepared from the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) at pH 8.0. After addition to these vesicles of the enzyme phospholipase D (PLD) from Streptomyces sp. AA586 at 40 degrees C, the terminal phosphate ester bond of POPC was hydrolyzed, yielding the negatively charged POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid) and the positively charged choline. While the reaction yield in the presence of 1 mM Ca2+ reached 100%, the yield was only approximately 68% in the absence of Ca2+. Furthermore, in the absence of Ca2+, the size of the vesicles did not change significantly with time upon PLD addition, as judged from turbidity, dynamic light scattering, and electron microscopy measurements. In the presence of 1 mM Ca2+, however, PLD addition resulted in vesicle aggregation, fusion, and precipitation, originating from the interaction of Ca2+ ions with the negatively charged phospholipids formed in the membranes. Vesicle fusion was monitored by using a novel fusion assay system involving vesicles containing entrapped trypsin and vesicles containing entrapped chymotrypsinogen A. After vesicle fusion, chymotrypsinogen A transformed into a-chymotrypsin, catalyzed by trypsin inside the fused vesicles. The alpha-chymotrypsin formed could be detected with benzoyl-L-Tyr-p-nitroanilide as a membrane permeable chymotrypsin substrate. The observed vesicle precipitation occurring after vesicle fusion in the presence of 1 mM Ca2+ was correlated with an increase of the main phase transition temperature, Tm, of POPA to values above 40 degrees C.     

A model for cation adsorption to clays and membranes

A cation adsorption model is presented and its recent applications are discussed. The model combines electrostatic equations with specific binding, and considers neutral and positively charged complexes between the negative surface sites and organic cations in a closed system. Extensions in the model account for dye aggregation in solution, and for the formation of solution complexes of inorganic cations, such as [M⁺⁺ Cl^–]⁺. The amounts of 45Ca²⁺ adsorbed to vesicles extracted from the plasma membranes of melon root cells could be adequately simulated and predicted. The binding coefficients determined for Ca²⁺, Na⁺, and Mg²⁺ are in the range of values previously deduced for binding to phospholipid components. Model calculations were applied to the test of hypotheses on the effect of salt stress on the growth of roots. The adsorption of monovalent organic cations to montmorillonite is characterized by binding coefficients that are at least six orders of magnitude larger than those of Na⁺, Mg²⁺, Ca²⁺, and Cd²⁺, or those of CdCl⁺ or CaCl⁺. Monovalent organic cations were found to adsorb 140–200% of the cation exchange capacity of the clay and to cause charge reversal. Deductions from adsorption results of acriflavin are consistent with those drawn from the application of other experimental methods. Preliminary results on the adsorption of divalent organic cations are presented. Agro-environmental applications of organo-clays are discussed.     

Study on size growth in the vesicle formation upon detergent removal from phospholipid/ detergent mixed micelles

When 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) was removed from the mixed CHAPS/EggPC micelles, large vesicles were prepared by dialysis or by slow step-by-step dilution, but small vesicles were prepared by fast one-step dilution. When sodium cholate was removed from the sodium cholate/EggPC micelles, small vesicles formed either by dialysis or by dilution; however, in the presence of 5 mM Ca²⁺ large vesicles were produced by dialysis, while small vesicles were prepared by dilution. The size growth was related to a detergent-induced fusion of the vesicles containing a large amount of detergent. Using spectrophotometry, quasielastic light scattering and freeze–fracture electron microscopy the fusion events were investigated both through the process of vesicle solubilization by adding detergent and through the process of vesicle formation by diluting a mixed micelle. The results suggest that a rapid CHAPS-induced fusion of the vesicles led to the large resultant vesicles and that no fusion of vesicles containing sodium cholate is responsible for the formation of small vesicles. Furthermore, the ultimate vesicle size related to rapid or slow detergent removal is dependent on the kinetic aspects of the fusion. Received: 19 August 1999 Accepted: 18 February 2000     

Participation of electrolyte cations in albumin adsorption onto negatively charged polymer latices

The participation of electrolyte cations in the adsorption of bovine serum albumin (BSA) onto polymer latices was investigated. The latices used were hydrophobic polystyrene (PS), and hydrophilic copolymers, i.e., styrene (St)/2-hydroxyethyl methacrylate(HEMA) copolymer [P(St/HEMA)] and styrene/acrylamide (AAm) copolymer [P(St/AAm)]. Three kinds of electrolyte cations (Na⁺, Ca²⁺, Mg²⁺) were used as the chloride. The amount of BSA adsorbed in every cation medium showed a maximum near the isoelectric point (iep, pH about 5) of the protein. The amounts of BSA adsorbed onto copolymer latices (except in the acidic pH region lower than the iep) were considerably smaller than that onto PS latex because of the steric repulsion and the decrease in the hydrophobic interaction between BSA and copolymer latices. In the acidic pH region, there was little difference in the amount of BSA adsorbed in every cation medium. However, in the pH region higher than the iep, the amounts of BSA adsorbed (particularly onto PS latex) in divalent cations (Ca²⁺ and Mg²⁺) media were relatively greater compared with that in a monovalent (Na⁺) one. This result was interpreted on the basis of the differences in such effects of electrolyte cations as dehydration power, suppression of the electrostatic repulsion, and binding affinity to BSA molecule. Ion Chromatographic estimation of the amounts of electrolyte cations captured upon BSA adsorption (in pH > 5) revealed that divalent cations were incorporated into the contact interface between the latex and BSA molecule so as to prevent the accumulation of anion charge and facilitate the protein adsorption.     

Surface properties of catalytic aluminas modified by alkaline-earth metal cations: A microcalorimetric and IR-spectroscopic study

Lewis acidic properties of transition aluminas whose surfaces have been doped with alkaline-earth metal cations (Ca²⁺ and Ba²⁺) were studied by means of the room temperature adsorption of carbon monoxide. The vibrational features of CO adsorbed at the surface of doped aluminas were investigated by IR spectroscopy in comparison with pure parent aluminas, while the quantitative and energetic features were studied by adsorption microcalorimetry. Various CO adspecies were found to form at the surface of both pure and doped-alumina, owing to the structural heterogeneity of the Al₂O₃ surface and to the presence of alkaline-earth metal cations. The surface heterogeneity was revealed by different v_co stretching frequencies, namely v_co≈2230, 2218 and 2205 cm⁻¹ for coordinatively unsaturated tetrahedral Al³⁺ cations in different crystallographic configurations, and v_co≈2186 and 2172 cm⁻¹ for coordinatively unsaturated Ca²⁺ and Ba²⁺ cations, respectively. Heats of adsorption of ≈80, 70 and 55 kJ/mol were assigned to the formation of Al³⁺/CO complexes, ≈45 kJ/mol for Ca²⁺/CO and ≈30 kJ/mol for Ba²⁺/CO complexes. The latter value was estimated through a correlation curve existing between v_co stretching frequencies and adsorption enthalpies. This correlation, already proposed in the past for CO adsorbed on non-d/d⁰/d¹⁰ metal cations, has been revisited and confirmed here, by including Al₂O₃ data for which an apparent lack of correlation between the two parameters was first observed. With respect to pure alumina, the population of Lewis acidic sites was found to be significantly depressed by the presence of alkaline-earth cus metal cations. These acidic sites are intrinsically weaker than tetrahedral cus Al³⁺ cations, as witnessed by smaller upward shifts of the v_co stretching frequencies with respect to CO gas and lower heats of adsorption, in accordance with expectations from the charge/ionic radius ratios. Ca²⁺ cations were found to compete in adsorbing CO with Al³⁺ cations more efficiently than the larger Ba²⁺ cations. In the case of CaO/Al₂O₃ systems outgassed at 1023 K, a thin surface layer of calcium aluminate, not detected by XRD or HRTEM, was suggested to form.     

Modulation of lysozyme charge influences interaction with phospholipid vesicles

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between charge state of the protein and its interaction with negatively charged phospholipid membranes chemical modifications of the proteins were carried out. Succinylation and carbodiimide modification was used to shift the isoelectric point of lysozyme to lower and higher pH values, respectively. The binding of the modified lysozyme to phospholipid vesicles prepared from phosphatidic acid (PA) was determined using microelectrophoresis and ultracentrifugation. At acidic pH of the solution all lysozyme species reduced the surface charges of PA vesicles. Succinylated lysozyme (succ lysozyme) reduced the electrophoretic mobility (EPM) to nearly zero, whereas native lysozyme and carboxylated lysozyme (carbo lysozyme) changed the surface charge to positive values. At neutral pH, the reduction of surface charges was less for carbo lysozyme and unmodified lysozyme. Succ lysozyme did not change the EPM. Unmodified and carbo lysozyme decreased the magnitude of EPM, but the whole complex was still negatively charged. The bound fraction of all modified lysozyme to PA vesicles at high lysozyme/PA ratios was nearly constant at acidic pH. At low lysozyme/PA ratios the extent of bound lysozyme is changed in the order carbo>unmodified>succ lysozyme. Increasing the pH, the extent of bound lysozyme to PA large unilamellar vesicles (LUV) is reduced, at pH 9.0 only 35% of carbo lysozyme, 23% of unmodified lysozyme is bound, whereas succ lysozyme does not bind at pH 7.4 and 9.0. At low pH, addition of all lysozyme species resulted in a massive aggregation of PA liposomes, at neutral pH aggregation occurs at much higher lysozyme/PA ratios. Lysozyme binding to PA vesicles is accompanied by the penetration of lysozyme into the phospholipid membrane as measured by monolayer techniques. The penetration of lysozyme into the monolayer was modulated by pH and ionic strengths. The interaction of lysozyme with negatively charged vesicles leads to a decrease of the phospholipid vesicle surface hydration as measured by the shift of the maximum of the fluorescence signal of a headgroup labeled phospholipid. The binding of bis-ANS as an additional indicator for the change of surface hydrophobicity is increased at low pH after addition of lysozyme to the vesicles. More hydrophobic patches of the lysozyme-PA complex are exposed at low pH. At low pH the binding process of lysozyme to PA vesicles is followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the vesicle aqueous content. The extent of lysozyme interaction with PA LUV at neutral and acidic pH is in the order carbo lysozyme>lysozyme>succ lysozyme.     

Organotin biocides XV: Modelling the interactions of triorganotins with cell membranes

The interaction of triorganotins with cell membranes has been examined using synthetic phospholipid membranes (vesicles). Electron microscopy and NMR methods indicate that the organotin is in a five-coordinate environment at the membrane surface, and is associated weakly with the phosphate headgroups of the vesicle components. Both trimethyl- and tributyl-tin cause extensive modifications to the vesicles including fusion, aggregation, blebbing and total rupture, these effects being initiated at concentrations as low as 25 μmol dm^?3.     

Multifrequency ESR and ENDOR studies of ionomers

We describe methods for determining the local environment of cations and the process of ionic clustering in ionomers, using electron magnetic resonance spectroscopy. The distance between Cu²⁺ cations in perfluorinated membranes (Nafion) containing terminal sulfonic groups and swollen by water has been deduced from an analysis of ESR spectra at L (1.25 GHz), S (2.36 GHz) and X (9.36 GHz) bands, in membranes containing cupric ion concentrations in the range 1–30 percent of the total amount needed to fully neutralize the pendant acid groups. At higher cation concentrations ESR spectra indicate the presence of aggregated cations. The intercation distance determination is based on the simulation of spectra from isolated cations using distribution widths δg₁₁ and δA₁₁ and extraction of the residual width ΔH^R, which is due to dipolar interactions. No aggregation is detected in membranes swollen by less polar solvents such as methanol, dimethylformamide (DMF) and tetrahydrofuran (THF); these results are in contrast to SAXS experiments in membranes swollen by methanol, which exhibit the “ionic peak”. Cu²⁺-Cu²⁺ and Ti³⁺-Ti³⁺ dimers have been detected in Nafion swollen by water, methanol, DMF and THF, and have been characterized by an analysis of the spin-forbidden half-field Δm_s=2 transition, and by computer simulations. The intercation distance in the cupric dimers, deduced from the intensity ratio of the Δm_s=2 and Δm_s=1 dimer transitions, is 5.0±0.2 Å. A model for the dimer has been proposed, which explains the crosslinking of the polymer chains by the metal cations. ENDOR signals from ¹H, ²H and ¹⁹F nuclei have been detected in Nafion neutralized by Ti³⁺. The ENDOR results allow determination of the local environment of the paramagnetic cations, to a distance of ∼10 Å.     

Affinity capillary electrophoresis employed for determination of stability constants of antamanide complexes with univalent and divalent cations in methanol

Affinity capillary electrophoresis (ACE) and pressure‐assisted ACE were employed to study the noncovalent molecular interactions of antamanide (AA), cyclic decapeptide from the deadly poisonous fungus Amanita phalloides, with univalent (Li⁺, Na⁺, K⁺, and NH₄⁺) and divalent (Mg²⁺ and Ca²⁺) cations in methanol. The strength of these interactions was quantified by the apparent stability constants of the appropriate AA‐cation complexes. The stability constants were calculated using the nonlinear regression analysis of the dependence of the effective electrophoretic mobility of AA on the concentration of the above ions in the BGE (methanolic solution of 20 mM chloroacetic acid, 10 mM Tris, pH_MeOH 7.8, containing 0–50 mM concentrations of the above ions added in the form of chlorides). Prior to stability constant calculation, the AA effective mobilities measured at actual temperature inside the capillary and at variable ionic strength of the BGEs were corrected to the values corresponding to the reference temperature of 25°C and to the constant ionic strength of 10 mM. From the above ions, sodium cation interacted with AA moderately strong with the stability constant 362 ± 16 L/mol. K⁺, Mg²⁺, and Ca²⁺ cations formed with AA weak complexes with stability constants in the range 37–31 L/mol decreasing in the order K⁺ > Ca²⁺ > Mg²⁺. No interactions were observed between AA and small Li⁺ and large NH₄⁺ cations.     

Capillary ion electrophoresis-capacitively coupled contactless conductivity detection of inorganic cations in human saliva on a polyvinyl alcohol-coated capillary

Capillary ion electrophoresis–capacitively coupled contactless conductivity detection (CIE-C4D) with a polyvinyl alcohol chemically coated capillary (PVA capillary) was used to analyze inorganic cations (Na⁺, K⁺, NH₄⁺, Mg²⁺, and Ca²⁺) commonly found in human saliva. The PVA capillary, which was made by our laboratory, minimized electro-osmotic flow in the wide pH range of the background electrolyte (BGE), and the PVA layer adsorbed to capillary wall did not affect the conductimetric background level. In this study, we determined an optimized BGE of 30 mM lactic acid/histidine plus 3 mM 18-crown-6 for the CIE-C4D system using the PVA capillary, which could simultaneously improve the separation of Mg²⁺ and Ca²⁺ from Na⁺ and that of K⁺ from NH₄⁺. This system obtained highly reproducible separation of cations in human saliva samples within 8 min at 20 kV without deprotonation. The quantifiability of cations in human saliva samples on the CIE-C4D system was demonstrated through identification by ion chromatography with satisfactory results.     

Application of pure silica gel as cation-exchange stationary phase in lon chromatography with indirect photometric detection for common mono-and divalent cations using aromatic monoamines as eluents

Summary A pure silica gel (Pia Seed 5S-60-SIL), synthesized by the hydrolysis of pure tetraethoxysilane [Si(OCH₂CH₃)₄], was applied as a cation-exchange stationary phase in ion chromatography with indirect photometric detection for common mono-and divalent cations (Li⁺, Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺) using various protonated aromatic monoamines (tyramine [4-(2-aminethyl) phenol], benzylamine, phenylethylamine, 2-methylpyridine and 2,6-dimethylpyridine) as eluet ions. When using 0.75 mM tyramine-0.25 mM oxalic acid-1.5 mM 18-crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) at pH 5.0 as the eluent, excellent simultaneous separation and highly sensitive detection at 275 nm for these mono-and divalent cations were achieved on the Pia Seed 5S-60-SIL column (150×4.6 mm I.D.) in 20 min.     

Stability of phospholipid vesicles studied by asymmetrical flow field-flow fractionation and capillary electrophoresis

The stability of zwitterionic phosphatidylcholine vesicles in the presence of 20 mol% phosphatidyl serine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI), and diacylphosphatidyl glycerol (PG) phospholipid vesicles, and cholesterol or calcium chloride was investigated by asymmetrical flow field-flow fractionation (AsFlFFF). Large unilamellar vesicles (LUV, diameter 100 nm) prepared by extrusion at 25 °C were used. Phospholipid vesicles (liposomes) were stored at +4 and −18 °C over an extended period of time. Extruded egg yolk phosphatidylcholine (EPC) particle diameters at peak maximum and mean measured by AsFlFFF were 101 ± 3 nm and 122 ± 5 nm, respectively. No significant change in diameter was observed after storage at +4 °C for about 5 months. When the storage period was extended to about 8 months (250 days) larger destabilized aggregates were formed (172 and 215 nm at peak maximum and mean diameters, respectively). When EPC was stored at −18 °C, large particles with diameters of 700–800 nm were formed as a result of dehydration, aggregation, and fusion processes. In the presence of calcium chloride, EPC alone did not form large aggregates. Addition of 20 mol% of negatively charged phospholipids (PS, PA, PI, or PG) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles increased the electrostatic interactions between calcium ion and the vesicles and large aggregates were formed. In the presence of cholesterol, large aggregates of about 250–350 nm appeared during storage at +4 and −18 °C for more than 1 day.

The effect of liposome storage temperature on phospholipid coatings applied in capillary electrophoresis (CE) was studied by measuring the electroosmotic flow (EOF). EPC coatings with and without cholesterol, PS, or calcium chloride, prepared from liposomes stored at +25, +4, and −18 °C, were studied at 25 °C. The performances of the coatings were further evaluated with three uncharged compounds. Only minor differences were observed between the same phospholipid coatings, showing that phospholipid coatings in CE are relatively insensitive to storage at +25, +4 °C or −18 °C.     

Simultaneous separation of common mono- and divalent cations on silica gels modified with aluminium and zirconium

The modification of silica gel with aluminium and zirconium can be used for the preparation of advanced silica-based cation-exchange stationary phases for use in ion chromatography with conductimetric detection (IC-CD) for cations. Silica gels modified with aluminium (Al-silica) and zirconium (Zr-silica) act as cation-exchangers under strongly acidic conditions. Highly sensitive indirect conductimetric detection and excellently simultaneous separation for common mono- and divalent cations (Li⁺, Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺) can be achieved on the Al-silica and Zr-silica columns in IC-CD by using acidic eluents containing 15-crown-5 (1,3,7,10,13-pentaoxacyclopentadecane). The Al-silica and Zr-silica can also be applied successfully as cation-exchange stationary phases in ion-exclusion chromatography for the separation of various aliphatic and benzenecarboxylic acids.     

PHOTOPHYSICS OF THE FLUORESCENT Ca2+ INDICATOR QUIN-2

The photophysics of the complex forming reaction between Quin-2 and Ca²⁺ were investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with EGTA as Ca²⁺ buffer: k₀₁= 8.6 times 10⁸ s^?1, k₂₁= 1 times 10¹¹M^?1 s^?1, k₀₂= 8.8 times 10⁷ s^?1, k₁₂= 4 times 10⁴ s^?1. k₀₁ and k₀₂ denote the respective deactivation rate constants of the Ca²⁺ free and bound forms of Quin-2 in the excited state. The constant k₂₁ represents the second-order rate constant of binding of Ca²⁺ and Quin-2 in the excited state while k₁₂ is the first-order rate constant of dissociation of the excited Ca²⁺:Quin-2 complex. From the estimated values of k₁₂ and k₂₁ the dissociation constant K_d* in the excited state was calculated. It was found that pK_d* (6.4) is slightly smaller than pK_d (7.2). There was no interference of the excited-state complex forming reaction with the determination of K_d. Intracellular Ca²⁺ concentrations can thus accurately be determined from fluorometric measurements using Quin-2 as Ca²⁺ indicator.     

Macromolecule-to-Amphiphile Conversion Process of a Polyoxometalate–Polymer Hybrid and Assembled Hybrid Vesicles

We report our findings on the macromolecule-to-amphiphile conversion process of a polyoxometalate–polymer hybrid and the assembled hybrid vesicles formed by aggregation of the hybrid amphiphile. The polyoxometalate–polymer hybrid is composed of a polyoxometalate (POM) cluster, which is covered by five tetrabutylammonium (Bu₄N⁺) countercations, and a polystyrene (PS) chain. Through a cation-exchange process the Bu₄N⁺ countercations can be replaced by protons to form a hybrid amphiphile composed of a hydrophilic, protonated POM cluster and a hydrophobic PS chain. By implementing a directed one-dimensional diffusion and analyzing the diffusion data, we confirmed that the diffusion of solvated protons rather than macromolecules or aggregates is the key factor controlling the conversion process. Once the giant hybrid amphiphiles were formed, they immediately assembled into kinetically favored vesicular aggregates. During subsequent annealing these vesicular aggregates were transformed into thermodynamically stable vesicular aggregates with a perfect vesicle structure. The success in the preparation of the POM-containing hybrid vesicles provides us with an opportunity of preparing POM-functionalized vesicles.