Hydrostatic pressure induces hydrocarbon chain interdigitation in single-component phospholipid bilayers

By use of neutron diffraction for structural analysis, the temperature-pressure phase diagrams of several fully hydrated single-component phospholipid bilayers have been explored up to hydrostatic pressures of 2 kbars. The gel to liquid-crystalline phase transition temperature Tm increases linearly with pressure over a 10(-3)-2 kbar range in accordance with the Clausius-Clapeyron relationship giving dTm/dP values of 23.0 degrees C/kbar for 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 28.0 degrees C/kbar for 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The so-called pretransition was not observed in the isothermal pressure experiments, suggesting that no appreciable volume change occurs at this transition. These results are in good agreement with those reported using other techniques. In addition, at pressures higher than the isothermal liquid-crystalline to gel transition pressure, a new pressure-induced phase transition was observed for DPPC and DSPC in which the hydrocarbon chains from apposing monolayers become interdigitated with the chains occupying a cross-sectional area approximately equal to 5% less than in the gel phase. The temperature-pressure phase diagrams show the gel-interdigitated phase boundaries to be highly curved and the minimum pressure at which interdigitation occurs to decrease with increasing hydrocarbon chain length.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Comparative study of the gel phases of ether- and ester-linked phosphatidylcholines

Calorimetric, X-ray diffraction, and 31P nuclear magnetic resonance (NMR) studies of aqueous dispersions of 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) gel phases at low temperatures (-60 to 22 degrees C) show thermal, structural, and dynamic differences when compared to aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) gel phases at corresponding temperatures. Differential scanning calorimetry of DHPC dispersions demonstrates a reversible, low-enthalpy "subtransition" at 4 degrees C in contrast to the conditionally reversible, high-enthalpy subtransition observed at 17 degrees C for annealed DPPC bilayers. X-ray diffraction studies indicate that DHPC dispersions form a lamellar gel phase with dav congruent to 46 A both above and below the "subtransition". It is suggested that the reduced dav observed for DHPC (46 A as compared to 64 A in DPPC) is due to an interdigitated lamellar gel phase which exists at all temperatures below the pretransition at 35 degrees C. 31P NMR spectra of DHPC gel-phase bilayers show an axially symmetric chemical shift anisotropy powder pattern which remains sharp down to -20 degrees C, suggesting the presence of fast axial diffusion. In contrast, 31P spectra of DPPC bilayers indicate this type of motion is frozen out at approximately 0 degrees C.     

Ether phosphatidylcholines: comparison of miscibility with ester phosphatidylcholines and sphingomyelin, vesicle fusion, and association with apolipoprotein A-I

Nonhydrolyzable matrices of ether-linked phosphatidylcholines (PCs) and sphingomyelin have been used to study the mechanism of action of lipolytic enzymes. Since ether PCs, sphingomyelin, and ester PCs vary in the number of hydrogen bond donors and acceptors in the carbonyl region of the bilayer, we have examined several physical properties of ether PCs and sphingomyelin in model systems to validate their suitability as nonhydrolyzable lipid matrices. The intermolecular interactions of ether PCs with ester PCs, sphingomyelin, and cholesterol were investigated by differential scanning calorimetry. Phase diagrams constructed from the temperature dependence of the gel to liquid-crystalline phase transition of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether) and 1,2-O-ditetradecyl-sn-glycero-3-phosphocholine (DMPC-ether) with both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) demonstrated complete lipid miscibility in the gel and liquid-crystalline phases. Additionally, phase diagrams of egg yolk sphingomyelin (EYSM) with DMPC or DMPC-ether and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-O-dioctadecyl-sn-glycero-3-phosphocholine (DSPC-ether) demonstrated no major differences in miscibility of EYSM in ester and ether PCs. The effect of 10 mol % cholesterol on the thermal transitions of mixtures of ester and ether PCs also indicates little preference of cholesterol for either lipid. The fusion of small single bilayer vesicles of DMPC, DMPC-ether, DPPC, and DPPC-ether to larger aggregates as determined by gel filtration indicated that the ester PC vesicles were somewhat more stable.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-pressure infrared spectroscopy of ether- and ester-linked phosphatidylcholine aqueous dispersions.

Infrared spectra of aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and its ether-linked analogue, 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC), were measured in a diamond anvil cell at 28 degrees C as a function of pressure up to 20 kbar. Although these two lipids differ only in the linkages to the saturated hydrocarbon chains, significant differences were observed in their barotropic behavior. Most notable were the magnitudes of the pressure-induced correlation field splittings of the methylene scissoring and rocking modes, and the relative intensities of the corresponding component bands. In the case of the scissoring mode, not only can the correlation field component band be resolved at a lower pressure in DHPC (1.2 kbar, as compared with 2.2 kbar in DPPC), but the initial magnitude of the correlation field splitting in DHPC, particularly less than 9 kbar, is significantly greater than that observed in DPPC. These differences are attributed to the presence of an interdigitated lamellar gel phase in DHPC. At all pressures where the correlation field component band delta'CH2 can be resolved, the relative peak height/intensity ratio R = I delta'/I delta is greater in DPPC than in DHPC, suggesting that this parameter may be useful as a test of interdigitation.     

Effect of 2,4-dichlorophenol on DPPC/water liposomes studied by X-ray and freeze-fracture electron microscopy

The effect of 2,4-dichlorophenol (DCP) was studied on the fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)--water liposomes. The structure and the thermotropic phase behaviour of the liposomes was examined in the presence of DCP (DCP/DPPC molar ratio, varied from 2x10(-2) up to 1) using small- and wide-angle X-ray scattering (SAXS, WAXS) and freeze-fracture electron microscopy. The structural behaviour of the DPPC/DCP/water system was strongly dependent on the concentration of the DCP. In the pretransition range the DCP molecules (at 2x10(-2) DCP/DPPC molar ratio) induced the interdigitated phase beside the parent (gel and rippled gel) phases, locally which can be form at higher DCP concentration. When the DCP/DPPC molar ratio was increased the pretransition disappeared and the main transition was shifted to lower temperatures. In the molar ratio range from 2x10(-1) up to 5x10(-1), a coexistence of different phases was observed in the wide temperature range from 20 up to 40 degrees C. With a further increase of the DCP/DPPC molar ratio (6x10(-1) to 1) only the interdigitated gel phase occurred below 25 degrees C. A schematic phase diagram of DPPC/DCP/water system was constructed to summarise the results.     

High-pressure 31P NMR study of dipalmitoylphosphatidylcholine bilayers.

High-pressure 31P NMR was used for the first time to investigate the effects of pressure on the structure and dynamics of the phosphocholine headgroup in pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar aqueous dispersions and in DPPC bilayers containing the positively charged form of the local anesthetic tetracaine (TTC). The 31P chemical shift anisotropies, delta sigma, and the 31P spin-lattice relaxation times, T1, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for both pure DPPC and DPPC/TTC bilayers. This pressure range permitted us to explore the rich phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as gel I (P beta'), gel II (L beta'), gel III, gel IV, gel X, and the interdigitated, Gi, gel phase. For pure DPPC bilayers, pressure had an ordering effect on the phospholipid headgroup within the same phase and induced an interdigitated Gi gel phase which was formed between the gel I (P beta') and gel II (L beta') phases. The 31P spin-lattice relaxation time measurements showed that the main phase transition (LC to gel I) was accompanied by the transition between the fast and slow correlation time regimes. Axially symmetric 31P NMR lineshapes were observed at pressures up to approximately 3 kbar but changed to characteristic axially asymmetric rigid lattice lineshapes at higher pressures (3.1-5.1 kbar).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pressure studies on two hydrated phospholipids--1,2-dimyristoyl-phosphatidylcholine and 1,2-dipalmitoyl-phosphatidylcholine

We present results of studies on the effect of pressure on phase transitions in 1,2-dimyristoyl-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-phosphatidylcholine (DPPC) dispersed in excess water. The P-T diagram of hydrated DMPC shows a Gel III-Gel II-Gel I triple point at 3.5 kbar, 41 degrees C, the Gel III phase being obtained by annealing the sample at high pressure for several hours. In the case of DPPC, a pressure induced phase (X) appears between the Gel II and Gel I phases at approximately 0.93 kbar. With increasing pressure the temperature range of the X phase increases at the expense of that of the Gel I phase until finally at 2.87 kbar, the latter is completely suppressed. The P-T diagram of water-rich DPPC thus has 2 triple points, the Gel II-X-Gel I triple point at 0.93 kbar, 42.5 degrees C and the X-Gel I-liquid crystal triple point at 2.87 kbar, 98.5 degrees C. A pressure induced Gel III-Gel II transition is also observed in DPPC in the pressure range of 1.7-3 kbar.     

Interaction of phospholipid dispersions with water-soluble porphyrins as monitored by their Raman temperature profiles

Raman scattering spectra of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) dispersions, mixed with water-soluble porphyrins, i.e. cationic copper(II)-5,10,15,12-tetrakis(4-N-methylpyridyl) and anionic silver(II)-5,10,15,20-tetrakis(4-carboxyphenyl)porphyrins, were measured in the 2800-3100 cm(-1) C-H stretching vibration region as a function of the temperature within the 5-55 degrees C range. Temperature profiles of Raman data were constructed from a quantitative data treatment based on factor analysis. This method is shown to be more efficient than the commonly used approach employing peak intensity ratios. Parameters of the gel phase to liquid crystal phase transition determined from Raman temperature profiles were used to monitor the porphyrin influence on DPPG and DPPC structures. Both negatively and positively charged porphyrins significantly perturb DPPC and DPPG dispersions, causing significant downshift of the transition temperature and broadening of the transition region. Water-soluble porphyrins are assumed to set at the outside part of phospholipid dispersions and interact via coulombic forces with charged lipid heads. For the cationic CuTMPyP, the strongest effect has been observed for negatively charged DPPG. In contrast, anionic AgTPPC4 has been found to interact more efficiently with DPPC possessing both positive and negative charges.     

A calorimetric study of the thermotropic behavior of pure sphingomyelin diastereomers

The phase-transition properties of sphingomyelins were investigated in detail with totally synthetic, chemically and stereochemically pure (2S,3R)-(N-stearoylsphingosyl)-1-phosphocholine (D-erythro-C18-SPM) (1) and the corresponding 2S,3S isomer (L-threo-C18-SPM) (2). Heating scans of an unsonicated dispersion of 1 right after hydration showed a main transition (I) at 44.7 degrees C (delta H = 6.8 kcal/mol). Upon incubation at 20-25 degrees C a second transition (II) appeared at 36.0 degrees C (delta H = 5.7 kcal/mol). The two gel phases were designated as G alpha and G beta phases, respectively. The G beta phase was also metastable and relaxed to a third gel phase (G gamma) upon incubation below 10 degrees C. Conversion of the G gamma phase to the liquid-crystalline phase occurred via two new endotherms at 33.4 degrees C (2.6 kcal/mol) (III) and 43.6 degrees C (8.0 kcal/mol) (IV) as well as a main transition at 44.7 degrees C (9.5 kcal/mol). Possible interpretations have been proposed to account for the observed phase transitions. The L-threo isomer 2 showed similar thermotropic behavior to dipalmitoylphosphatidylcholine (DPPC): a "main transition" at 44.2 degrees C (6.0 kcal/mol), a "pretransition" at 43.1 degrees C (1.8 kcal/mol), and upon incubation at 7 degrees C for 2 weeks, a very broad "subtransition" at ca. 35 degrees C. The results are substantially different from previous studies of sphingomyelins using mixtures of stereoisomers. Mixing of 1 with 2, 1 with DPPC, and 2 with DPPC removed the metastability of the gel phase and resulted in a single transition.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 degrees C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L alpha-L beta') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 degrees C commencing at the calorimetric phase transition temperature.     

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.     

Bilayer interactions of ether- and ester-linked phospholipids: dihexadecyl- and dipalmitoylphosphatidylcholines

Mixed phospholipid systems of ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) and ester-linked 1,2-dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry and X-ray diffraction. At maximum hydration (60 wt % water), DHPC shows three reversible transitions: a main (chain melting) transition, TM = 44.2 degrees C; a pretransition, TP = 36.2 degrees C; and a subtransition, TS = 5.5 degrees C. DPPC shows two reversible transitions: TM = 41.3 degrees C and TP = 36.5 degrees C. TM decreases linearly from 44.2 to 41.3 degrees C as DPPC is incorporated into DHPC bilayers; TP exhibits eutectic behavior, decreasing sharply to reach 23.3 degrees C at 40.4 mol % DPPC and then increasing over the range 40-100 mol % DPPC; TS remains constant at 4-5 degrees C and is not observed at greater than 20 mol % DPPC. At 50 degrees C, X-ray diffraction shows a liquid-crystalline bilayer L alpha phase at all DHPC:DPPC mole ratios. At 22 degrees C, DHPC shows an interdigitated bilayer gel L beta phase (bilayer periodicity d = 47.0 A) into which approximately 30 mol % DPPC can be incorporated. Above 30 mol % DPPC, a noninterdigitated gel L beta' phase (d = 64-66 A) is observed. Thus, at T greater than TM, DHPC and DPPC are miscible in all proportions in an L alpha bilayer phase. In contrast, a composition-dependent gel----gel transition between interdigitated and noninterdigitated bilayers is observed at T less than TP, and this leads to eutectic behavior of the DHPC/DPPC system.     

Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) > DPPC > DMPC > SOPC > POPC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced dehydration of liposomal membranes provides the driving force for the segregation of the pyrene lipid.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the stability of the ripple phase in the DPPC/PLPC/water ternary system

The effect of incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine (PLPC) on the structure of the P_β′ ripple mesophase in aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been studied by differential scanning calorimetry (DSC) and scanning dilatometry (SD). For samples containing 34 wt. % ²H₂O and 0–15 wt. % PLPC, a pretransition was observed by DSC. The pretransition disappears at 15 wt. % PLPC. The behavior of thermodynamic functions at the pretransition and main transition gives new insights on the structural changes produced by PLPC on bilayers of DPPC.     

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase

A new thermotropic phase transition, at ?30°C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at ?60°C. The thermotropic phase transition at ?30°C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30°C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.     

Pentachlorophenol-induced change of zeta-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.     

Diazeniumdiolate reactivity in model membrane systems.

The effect of small unilamellar phospholipid vesicles on the acid-catalyzed dissociation of nitric oxide from diazeniumdiolate ions, R(1)R(2)N[N(O)NO](-), [1: R(1)=H(2)N(CH(2))(3)-, R(2)=H(2)N(CH(2))(3)NH(CH(2))(4)-; 2: R(1)=R(2)=H(2)N(CH(2))(3)-; 3: R(1)=n-butyl-, R(2)=n-butyl-NH2+(CH(2))(6)-; 4: R(1)=R(2)=nPr-] has been examined at pH 7.4 and 37 degrees C. NO release was catalyzed by anionic liposomes (DPPG, DOPG, DMPS, POPS and DOPA) and by mixed phosphatidylglycerol/phosphatidylcholine (DPPG/DPPC and DOPG/DPPC) covesicles, while cationic liposomes derived from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic liposome DMPC did not significantly affect the dissociation rates of the substrates examined. Enhancement of the dissociation rate constant in DPPG liposome media (0.010M phosphate buffer, pH 7.4, 37 degrees C) at 10mM phosphoglycerol levels, ranged from 37 for 1 to 1.2 for the anionic diazeniumdiolate 4, while DOPA effected the greatest rate enhancement, achieving 49-fold rate increases with 1 under similar conditions. The observed catalysis decreases with increase in the bulk concentration of electrolytes in the reaction media. Quantitative analysis of catalytic effects has been obtained through the application of pseudo-phase kinetic models and equilibrium binding constants at different liposome interfaces are compared. The stoichiometry of nitric oxide release from 1 and 2 in DPPG/DPPC liposome media has been obtained through oxyhemoglobin assay. DPPG=1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DOPG=1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DMPS=1,2-dimyristoyl-sn-glycero-3-[phospho-l-serine], POPS=1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine], DOPA=1,2-dioleoyl-sn-glycero-3-phosphate; DPPC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DMPC=1,2-dimyristoyl-sn-glycero-3-phosphocholine, DOTAP=1,2-dioleoyl-3-trimethylammonium-propane.