Pavel Smejtek

Electrical conductivity in lipid bilayer membranes induced by pentachlorophenol

Electrical conductivity induced in thin lipid bilayer membranes by pentachlorophenol has been studied. The membranes were formed from phosphatidyl choline, phosphatidyl ethanolamine, or phosphatidyl glycerol and various amounts of cholesterol. The position and the magnitude of the maximum of the conductivity vs. pH curve depend on the type of lipids and cholesterol content. At low pentachlorophenol concentrations and low pH the concentration dependence of conductivity is quadratic and becomes linear at higher pH. Above 10(-5) M of pentachlorophenol the concentration dependence of the membrane conductivity tends to saturate. Presence of pentachlorophenol enhances membrane transport of nonactin-K+ complex. Increase of cholesterol content increases pentachlorophenol induced conductivity in all membranes and shifts the conductivity toward lower pH. For phosphatidyl choline the largest rate of change of membrane conductivity with cholesterol occurs at 1:1 phospholipid to cholesterol molar ratio. Pentachlorophenol is found to be a class II uncoupler and the experimental results are consistent with the hypothesis that the membrane permeable species are dimers formed by combination of neutral and dissociated pentachlorophenol molecules. Several schemes of membrane conduction, including dimer formation in the aqueous phase as well as at the membrane-water interface have been considered. Arguments are given in favor of the formation of dimers within the membrane surface.

Adsorption of ruthenium red to phospholipid membranes

We have measured the distribution of the hexavalent ruthenium red cation (RuR) between water and phospholipid membranes, have shown the critical importance of membrane negative surface charge for RuR binding, and determined the association constant of RuR for different phospholipid bilayers. The studies were performed with liposomes made of mixtures of zwitterionic L-alpha-phosphatidylcholine (PC), and one of the negatively charged phospholipids: L-alpha-phosphatidylserine (PS), L-alpha-phosphatidylinositol (PI), or L-alpha-phosphatidylglycerol (PG). Lipid composition of PC:PX membranes was 1:0, 19:1, 9:1, and 4:1. Liposomes were processed using freeze-and-thaw treatment, and their size distribution was characterized by light scattering and electron microscopy. Experimental distribution isotherms of RuR obtained by ultracentrifugation and spectrophotometry can be reproduced with the Langmuir-Stern-Grahame model, assuming that RuR behaves in the diffuse double layer as an ion with effective valency < 6. In terms of this model, PC-PS, PC-PI, and PC-PG membranes were found to be electrostatically equivalent and the intrinsic association constants of RuR were obtained. RuR has highest affinity to PS-containing membranes; its association constant for PC-PI and PC-PG membranes is about 5 times smaller than that for PC-PS membranes. From the comparison of RuR binding to mixed negatively charged phospholipid membranes and RuR binding to sarcoplasmic reticulum (SR), we conclude that the low-affinity RuR binding sites may indeed be associated with the lipid bilayer of SR.

Distribution of hydrophobic ionizable xenobiotics between water and lipid membranes: Pentachlorophenol and pentachlorophenate. A comparison with octanol-water partition

We have studied distribution of pentachlorophenol (PCP)—a major environmental pollutant—between egg-phosphatidylcholine (egg-PC) membranes and water. The objectives were (1) to compare the membrane-water partition of the un-ionized (HA) and ionized (A) PCP, and (2) to establish similarities and differences between the partition of PCP into lipid membranes and into octanol. The studies were made with egg-PC liposomes. It is shown that the distribution isotherms can be understood in terms of the Langmuir-Stern-Grahame adsorption model. The model is applicable to both the HA and A species; it takes into account the electrostatic interactions at the membrane-water interface charged by the adsorbed pentachlorophenate. Relationships between the membrane surface adsorption and bulk partition characteristics were presented and used to relate the partition of PCP into egg-PC membranes to those for octanol-water systems. Results (egg-PC membranes): bulk distribution coeff. γHA=2.9×105, γA=1.6×104, association constant KmHA=2.9×105 M−1, KmA=0.7×105 M−1, adsorption site area PsHA=0.6 nm2, PsA=3.5 nm2, and linear partition coeff. βmHA=550 μm, βmA=30 μm. Comparable to γHA and γA for octanol-water are Pow(HA)≈1.3×105 and Pow(A)≈30. The major difference is in the distribution of ionized PCP which is several hundred times greater for egg-PC membranes compared to octanol. The difference is associated with the properties of the membrane-water interface.

Adsorption to dipalmitoylphosphatidylcholine membranes in gel and fluid state: pentachlorophenolate, dipicrylamine, and tetraphenylborate

UNLABELLED We measured the dependence of electrophoretic mobility of dipalmitoylphosphatidylcholine (DPPC) vesicles on the aqueous concentration of negatively charged ions of pentachlorophenol (PCP), dipicrylamine (DPA), and tetraphenylborate (TPhB). The objective was to determine how the physical state of hydrocarbon chains of lipids affects adsorption of lipophilic ions. The studies were done at 25 and 42 degrees C to determine adsorption properties of DPPC membrane in the gel and fluid state, respectively. From the analysis of zeta-potential isotherms in terms of Langmuir-Stern-Grahame model we obtained the association constant, K, the area of the adsorption site, Ps, and the linear partition coefficient, beta. RESULTS K, (x 10(4)M-1): K(gel): PCP (0.49 +/- 0.28), DPA (25 +/- 10), TPhB (31 +/- 10); K(fluid): PCP (4.5 +/- 0.9), DPA (74 +/- 21), TPhB (59 +/- 14); Ps, (nm2): Ps(gel): PCP (5.4 +/- 2.3), DPA (5.9 +/- 2), TPhB (5.0 +/- 1.7); Ps(fluid): PCP (4.5 +/- 0.4), DPA (5.2 +/- 0.4), TPhB (4.1 +/- 0.2); beta, (x 10(-5) m): beta(gel): PCP (0.15 +/- 0.09), DPA (7.1 +/- 0.3), TPhB (10 +/- 7); beta(fluid): PCP (1.7 +/- 0.3), DPA (24 +/- 7), TPhB (24 +/- 6). It was interesting to find that the adsorption site area for PCP, DPA, and TPhB were very similar for both the gel and fluid membranes; also, the areas were independent of the size and molecular structure of the adsorbing species. Using a simple discrete charge model the adsorption site areas for all species were consistent with a dielectric constant of 8-10 and with an ion adsorption depth of 0.4-0.6 nm below the water/dielectric interface. The delta delta G0 = delta G0(gel) - delta G0(fluid) was found to be about twice as large for PCP than for DPA and TPhB. This indicates that PCP will be significantly more adsorbed in the fluid and disordered regions of biomembranes, whereas the distribution of DPA and TPhB is expected to be relatively more even.

Dielectric properties of adsorption/ionization site of pentachlorophenol in lipid membranes.

The results of three complementary studies focused on characterization of the local environment of the common pesticide pentachlorophenol (PCP) adsorbed to phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes are reported. The effect of cholesterol (Chol) was examined. These studies included: Measurements of solvatochromic shifts of the ultraviolet absorption spectra of PCP in membranes and in polar non-hydrogen-bonding (a red shift) and hydrogen-bonding (a blue shift) solvents. Pi-pi transition energies were analyzed in terms of the dielectric cavity models of Onsager, Block-Walker, which includes dielectric saturation, and a soft dipole model of Suppan, which accounts for PCPs polarizability. The estimates of dielectric constant of the PCP adsorption site yielded 8.1-8.7 for the PC and 16.8-20.1 for PG membranes. Solvatochromic effects indicate hydrogen bonding between the membrane-bound ionized PCP molecule and water, which is enhanced by the presence of cholesterol. Determinations of the pKa of PCP adsorbed to PC, PG, PC/Chol, PG/Chol membranes and dissolved in dioxane-water solutions of a known dielectric constant. The pKa value of PCP adsorbed to membranes was always greater than the standard pKa value and it increased with the membranes negative charge. The pKa value sequence in 0.1 M KCl was 6.68 (PG), 6.32 (PG/Chol = 70:30 mole fractions), 5.97 (PC), and 5.75 (PC/Chol = 70:30). The intrinsic pKa values of PCP in membranes were 5.2-5.4 (PG) and 5.5-6.0 (PC). Estimates of the dielectric constant of PCPs ionization site in membranes yielded 10-22 (PC) and 27-37 (PG). Cholesterol facilitated the release of the hydrogen ion from membrane-bound PCP. Measurements of pH dependence of PCP-induced membrane electrical conductivity. pH values of conductivity maxima were always greater than the standard pKa of PCP, and their sequence corresponded to that of the pKa values of membrane-bound PCP. The anomalous properties of PCP as a Class 2 uncoupler are due to PCPs lipophilic character. In response to a low dielectric constant of the adsorption/ionization site, the physicochemical characteristics of PCP adsorbed to membranes are different from the standard values--a fact that needs to be taken into account in the development of models of PCPs toxicity.

Hydrophobicity and sorption of chlorophenolates to lipid membranes

We have studied sorption of ionized species of chlorophenols and pentahalophenols to lipid membranes using egg-phosphatidylcholine (egg-PC) vesicles and measuring their zeta-potential as a function of aqueous concentration of the phenolates. The zeta-potential isotherms can be understood in terms of a sorption model that is a combination of the Gouy-Chapman model of the electrical double layer at the membrane-water interface and the Langmuir model for sorption. Two intrinsic sorption parameters were determined: the linear partition coefficient beta m, which relates the membrane surface density of the phenolates to their aqueous concentration and the area of the adsorption site, Ps. The linear partition coefficient is the measure of the affinity of phenolates to the lipid membrane. It depends strongly on the molecular structure: 2,6-dichlorophenolate beta m = (0.45 +/- 0.08) x 10(-7); m; 3,5-dichlorophenolate beta m = (0.22 +/- 0.02) x 10(-6) m; 2,4,6-trichlorophenolate beta m = (0.63 +/-0.06) x 10(-6) m; 2,4,5-trichlorophenolate beta m = (0.11 +/- 0.01) x 10(-5) m; 2,3,5,6-tetrachlorophenolate beta m = (0.56 +/- 0.07) x 10(-5) m; 2,3,4,5-tetrachlorophenolate beta m = (0.55 +/- 0.06) x 10(-5) m; pentachlorophenolate beta m = (0.34 +/- 0.05) x 10(-4) m; pentafluorophenolate beta m = (1.00 +/- 0.13) x 10(-7) m and pentabromophenolate beta m = (0.19 +/- 0.04) x 10(-3) m. Ps was found to be independent of phenolate structure, Ps = 3.3 +/- 0.1 nm2. The membrane affinity of chlorophenolates was compared with the octanol-water partition coefficients of un-ionized chlorophenols. It was shown that the free energy of transfer of chlorophenolates from water into the lipid membrane can be divided into non-electrostatic and electrostatic contributions. The no-nelectrostatic contribution corresponds to the hydrophobicity parameter alpha = 3.94 +/- 0.0.08 kcal per nm2 of molecular surface area. The electrostatic contribution contains a term inversely proportional to the molecular radius of the phenolate ion which has the physical meaning of the work of transfer of the phenolate ion from water into the membrane. The polarity of the sorption region of egg-PC membranes is given in terms of the dielectric constant and was estimated to be 12.4 (range 10.5-13.4).

Induced hydrogen ion transport in lipid membranes as origin of toxic effect of pentachlorophenol in an alga

Abstract Pentachlorophenol (PCP) decreases the rate of carbon assimilation in the alga Selenastrum capricornutum . In parallel with the reduction of carbon assimilation in this alga there is a decrease of electrical resistance of lipid membranes and development of negative membrane surface charge. The experimental results suggest that PCP toxicity to algae is due to adsorption of negatively charged PCP ions at the membrane surface that act as carriers of hydrogen ion across the membrane. This protonophoretic action of PCP causes the decrease of membrane electrical resistance and the dissipation of hydrogen ion electrochemical potential gradients across cellular and subcellular membranes, which reduces the ability of algae to assimilate carbon.

The physicochemical basis of the membrane toxicity of pentachlorophenol: An overview☆

Abstract This report is concerned with the phenomenon of the membrane toxicity of pentachlorophenol (PCP)—a popular herbicide and wood preservative, and now one of the most widespread pollutants. We compare experimental data on membrane-PCP interactions from multiple studies. These data include membrane electrical conductivity, PCP toxicity, microelectrophoresis, and spectrophotometry. The membrane toxicity of PCP is associated with the PCP-induced hydrogen ion permeability of the lipid matrix of biomembranes. The onset of the toxic effect corresponds to the loss of membrane electrical resistance and the onset of measurable PCP adsorption. We show that electrophoresis of lipid vesicles can be effectively used to study PCP adsorption and that the PCP-membrane interaction can be described in terms of the Langmuir-Stern-Grahame model. We report how the solvatochromic shifts of the long wavelength UV absorption band of ionized PCP can be used to characterize the polarity of the PCP adsorption site on membranes. The dielectric constant of the site in phosphatidylcholine (PC) and in negatively charged phosphatidylglycerol (PG) membranes was found to be 8 and 20. The p K a of the dissociation of membrane-bound PCP is different from that in water (4.74). The largest p K a (6.7) was observed for PCP bound to negatively charged membranes. It was shown that when the membrane surface potential was taken into account, the intrinsic p K a s for the PC and PG membranes are approximately the same (5.1-5.6). This work illustrates the complementarity of studies done on lipid bilayer membranes and biological membranes.

Pentachlorophenol-induced change of ζ-potential and gel-to-fluid transition temperature in model lecithin membranes

We have determined zeta-potentials for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) membranes by measuring the electrophoretic mobility of multilayered vesicles and the temperatures of the gel-to-ripple-to-fluid phase transitions of sonicated vesicles by a photometric method. Some conclusions are: (1) The zeta-potentials of DMPC and DPPC vesicles become negative due to adsorption of ionized pentachlorophenol (PCP), (2) their magnitude changes, step-like, on gel-to-fluid transition and (3) the temperature of the step-like change in zeta-potential decreases with an increase in PCP concentration. (4) PCP exhibits a large effect on membrane structure: It induces an isothermal phase change from the ordered to disordered state, which is enhanced by monovalent salt in the aqueous phase. (5) Both ionized and unionized PCP decrease the melting phase transition temperature and abolish the pretransition, (6) the unionized species increases the melting transition width and (7) the ionized species is more potent in abolishing the pretransition. (8) The shorter chain lipid (DMPC) is more sensitive to the presence of PCP; the maximum decrease in delta Tt is 13 K (DMPC) and 7 K (DPPC) in the presence of ionized PCP. We have shown experimentally, by comparing the delta Tt from photometric studies with the density of adsorbed PCP derived from zeta-potential isotherms, that (9) the shift of the melting phase transition temperature increases linearly with the density of adsorbed PCP. (10) In contrast to membranes made of negatively charged lipids, the transition temperature of DMPC and DPPC membranes in the presence of PCP further decreases in the presence of monovalent salt. The salt effect is due to screening of the membrane surface leading to enhanced adsorption of ionized PCP and a depression in transition temperature. (11) It is shown that both the adsorption and the changes of gel-to-fluid phase transition temperature can be described in terms of the Langmuir-Stern-Grahame model and (12) proposed that future studies of membrane toxicity of PCP should be focused on its pH dependence.

AHA− heterodimer of a class-2 uncoupler: pentachlorophenol

AHA- heterodimers formed by association of neutral molecules of weak acid (HA) with its conjugate anion (A-) have been proposed to be the charged membrane-permeable species of class-2 uncouplers. Past attempts to extract and identify AHA- heterodimers failed. We have measured optical spectra of HA+A- (1:1) solutions of pentachlorophenol (PCP) in various solvents and in the presence of PC liposomes. Optical studies were supplemented by nuclear magnetic resonance measurements of HA+A- (1:1) solutions of PCP in dichloroethane to gain insight into the formation of AHA- species in lipid membranes. From these experiments, we found evidence for AHA- formation in non-hydrogen-bonding solvents, then reported the AHA- formation constant Kf and the molar absorptivity epsilon AHA-(lambda). Kf decreases with increasing dielectric constant, kappa, from 1210 +/- 130 M-1 for dichloroethane (kappa 10.7), to 340 +/- 34 M-1 for acetonitrile (kappa 37.5); Kf also decreases with increasing concentration of water. In hydrogen-bonding solvents, octanol (kappa 10.3) and methanol (kappa 33.5) and in liposomes, AHA- heterodimers are not observed optically. We estimate Kf for PCP in lipid bilayers from a combination of data on membrane electrical conductivity and surface density of adsorbed PCP. Our estimate for lipid bilayer, 0.005 < Kf < 0.5 M-1, is consistent with our inability to detect the AHA- species optically in liposome suspensions. We propose that penetration of water into the membrane inhibits formation of AHA- in lipid bilayers.