Influence of pH on phosphatidic acid multilayers. A rippled structure at high pH values.

The influence of pH on the structure of 1,2-(ditetradecyl)-phosphatidic acid was investigated by differential scanning calorimetry and freeze-fracture electron microscopy. At pH 13.5--14 (2.6 M K+), where phosphatidic acid has two negative charges, calorimetric scans show a small transition (pretransition) below the main phase transition temperature. Freeze-fracture studies of the same dispersions reveal regular band patterns (so-called ripples) in the plane of the bilayers, when the lipid is quenched from below the main phase transition temperature. This rippled structure is similar to the well-known rippled structure of phosphatidylcholines.     

The influence of metal cations and pH on the heat sensitivity of photosynthetic oxygen evolution and chlorophyll fluorescence in spinach chloroplasts

The heat-sensitivity of photosynthetic oxygen evolution of thylakoids isolated from spinach increases by increasing the pH above neutral value. The temperature for inactivation (transition temperature) is lowered from about 45° C (pH 6.0–7.4) to 33°C (pH 8.5). Similar results are obtained with intact chloroplasts. At pH 7.0 the transition temperature of washed thylakoids decreases by lowering the salt concentration below 20 mM with monovalent cations (Li⁺, Na⁺, K⁺) and below 3–4 mM with divalent cations (Mg²⁺, Ca²⁺, Sr²⁺). Illumination decreases the heat-sensitivity of oxygen evolution in intact chloroplasts, but even increases the heat-sensitivity in uncoupled chloroplasts. In intact chloroplasts the transition temperature of the heat-induced rise in chlorophyll fluorescence yield (F_o; see Schreiber and Armond 1978) decreases from 44° C to 38° C when the pH of the suspending medium is increased from 6.5 to 8.5. At 20° C, F_o is almost insensitive to pH (6.0–8.5). At 40° C, however, F_o is constant between 6.0 and 7.0, but strongly increases by increasing the pH above neutral value. The results are discussed in terms of a close relation between electrostatic forces at the thylakoid membrane and thermal sensitivity of photosynthetic apparatus. It is suggested that the heat-sensitivity of the photosystem II complex partially depends on the ionization state of fixed groups having alkaline pK. The packed volume of thylakoids suspended in a low salt medium increases when the temperature is increased above 30° C (pH 7.0) and above 20° C (pH 8.0), respectively. This result suggests a heat-induced increase in surface charge density of the thylakoid membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES morpholinoethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - TRICIN N-[tris(hydroxymethyl)-methyl] glycine     

Miscibility of phosphatidylcholine binary mixtures in unilamellar vesicles: Phase equilibria

Summary Miscibility among phospholipids with different lipid chain-lengths or with different head groups has attracted a number of research efforts because of its significance in biological membrane structure and function. The general consensus about the miscibility of phosphatidylcholines with varying lipid chainlengths appears to be that binary mixtures of phospholipids with a difference of two carbon atoms in the lipid chain mix well at the main phase transition. Miscibility between phosphatidylcholines with differences of four carbon atoms appears to be inconclusive. Previous reports on the phase transition of binary phospholipid mixtures are concerned mainly with multilamellar vesicles and are mostly limited to the main transition. In the present study, unilamellar vesicles were used and miscibility in binary systems between dimyristoyl-, dipalmitoyl- and distearoyl-phosphatidylcholines at pretransition, as well as main transition temperatures was evaluated by constructing phase diagrams. Two methods were used to monitor the phase transitions: differential scanning microcalorimetry and optical absorbance methods. The optical method has the advantage that unilamellar vesicles of dilute phospholipid concentrations can be used. The liquidus and solidus phase boundaries were determined by the onset temperature of heating and cooling scans, respectively, because the completion temperature of a phase transition has no meaning in binary solutions. Dimyristoyl- and distearoyl-phosphatidylcholines. where the difference in the, lipid chain-length is four carbon atoms, mixed well even at pretransition temperature.     

Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram

Small-angle neutron scattering has been used to study structural features of lamellar bilayer membranes of dimyristoylphosphatidylcholine (DMPC) and DMPC mixed with various amount of cholesterol. The studies were recorded at a fixed hydration level of 17% 2H2O, i.e. just below saturation. Bragg reflections gives information on the ripple structure and on the bilayer periodicity. The crystalline Lc phase, which was stabilized after long time storage at low temperature, exhibits major small angle scattering when cholesterol is mixed into the membrane. The intermediate P beta' gel-phase, which is characteristic by the rippled structure, is dramatically stabilized by the introduction of cholesterol. The ripple structure depends significantly both on the cholesterol content and on the temperature. At high temperatures, T greater than 15 degrees C, the inverse ripple periodicity varies basically linearly with cholesterol content, and approach zero (i.e. periodicity goes to infinite) at 20 mol% cholesterol, approximately. At lower temperatures the correlation is more complex. The data indicate additional phase boundaries below 2 mol% and at approx. 8 mol%. Secondary rippled structures are observed in the low temperature L beta'-phase for cholesterol content below approx. 8 mol%. The data gives detailed insight into the phosphatidylcholine cholesterol phase diagram, which is discussed on the basis of a simple model in which the cholesterol complexes are fixed to the defect stripes of the rippled structure.     

Detection of the metastable rippled gel phase in hydrated phosphatidylcholine by fluorescence spectroscopy

Steady-state and time-resolved emission spectroscopy of 1-anilinonaphthalene-8-sulfonic acid (ANS) have been used for characterization of the metastable rippled gel phase, Pbeta'(mst), formed in fully-hydrated dipalmitoylphosphatidylcholine (DPPC) upon cooling from the liquid crystalline phase Lalpha [Tenchov et al., Biophys. J. 56 (1989) 757]. The Pbeta'(mst) phase of DPPC clearly differs from the stable Pbeta' phase by increased (approximately 27%) ANS emission intensity, by enhanced (approximately 23%) average radiative rate constant, and by reduced (approximately 18%) non-radiative quenching rate constant. The fluorescence intensity peak at the Pbeta'-->Lalpha transition temperature is replaced by a large, reversible stepwise intensity drop at the Pbeta'(mst)-->Lalpha transition. No such effects have been found for dimiristoylphosphatidylcholine (DMPC) dispersions confirming previous results that DMPC does not form a Pbeta'(mst) phase. Since ANS is known to predominantly reside in the interfacial region, the observed effects indicate differences between the stable and metastable rippled phases in the organization and dynamics of their lipid/water interfaces. The data demonstrate that the metastable rippled phase manifests its appearance also through interactions with small molecules (ANS size approximately 8 A).     

Electrostatic interactions at charged lipid membranes. Measurement of surface pH with fluorescent lipoid pH indicators.

The 5-dimethylaminonapthalene-1-sulfonyl (dansyl) chromophore attached to the polar head groups of lipids has been used as a fluorescent lipoid pH indicator to evaluate the interfacial pH in lipid-water lamellar systems prepared from negatively charged lipids. The pH in the vicinity of the charged lipid bilayers is different from the pH of the bulk aqueous phase and the difference is a function of the electrolyte concentration in the aqueous phase and of the lipid packing in the bilayer. At a fixed electrolyte concentration in the aqueous phase, the observed interfacial pH is 0.6 to 0.7 pH units lower above the thermal phase transition of the lipid than it is below this temperature. A quantitative interpretation of the results is given on the basis of the Gouy-Chapman theory. The results indicate that the dansyl chromophore is located in front of the charged surface and its distance from this surface increases with a decrease in lipid packing.     

Structure and mechanical properties of giant lipid (DMPC) vesicle bilayers from 20 degrees C below to 10 degrees C above the liquid crystal-crystalline phase transition at 24 degrees C

We have used micromechanical tests to measure the thermoelastic properties of the liquid and gel phases of dimyristoylphosphatidylcholine (DMPC). We have found that the rippled P beta' phase is only formed when a vesicle is cooled to temperatures below the main acyl chain crystallization transition, Tc, under zero or very low membrane tension. We also found that the P beta' surface ripple or superlattice can be pulled flat under high membrane tension into a planar structure. For a ripple structure formed by acyl chains perpendicular to the projected plane, the projected area change that results from a flattening process is a direct measure of the molecular crystal angle. As such, the crystal angle was found to increase from about 24 degrees just below Tc to about 33 degrees below the pretransition. It was also observed that the P beta' superlattice did not form when annealed L beta' phase vesicles were heated from 5 degrees C to Tc; likewise, ripples did not form when the membrane was held under large tension during freezing from the L alpha phase. Each of these three procedures could be used to create a metastable planar structure which we have termed L*beta' since it is lamellar and plane-crystalline with acyl chains tilted to the bilayer plane. However, we show that this structure is not as condensed as the L beta' phase below 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel injectable pH and temperature sensitive block copolymer hydrogel

A novel pH and temperature sensitive block copolymer was prepared by adding pH sensitive moiety to temperature sensitive block copolymer. This block copolymer solution showed a reversible sol-gel transition by a small pH change in the range of pH 7.4-8.0 and also by the temperature change in the region of body temperature. The very precise molecular weight control of block copolymer and the prudential tuning of hydrophilic-hydrophobic balance were needed to control the phase diagram. This block copolymer solution forms a gel at 37 degrees C, pH 7.4 (human body). When the block copolymer solution is at room temperature and pH 8.0 as a sol state, both the temperature and pH change are needed for the gelation. This material can be employed as injectable carriers for hydrophobic drugs and proteins, etc. Gelation inside the needle can be prevented by an increase in the temperature during injection, because it does not change into the gel form with only increasing temperature. This material can be used for even a long guide catheter into the body. The block copolymer hydrogel which shows the sol-gel transition by the small pH change from pH 8.0 to pH 7.4 has merits in the delivery system for protein and cells which show cytotoxicity in acidic (below pH 6.5) or basic (above pH 8.5) conditions. This block copolymer system could be used as a template technology for injectable delivery systems.     

Morphological transitions of brain sphingomyelin are determined by the hydration protocol: ripples re-arrange in plane, and sponge-like networks disintegrate into small vesicles.

The phase transition of hydrated brain sphingomyelin occurs at around 35 degrees C, which is close to the physiological temperature. Freeze-fracture electron microscopy is used to characterize different gel state morphologies in terms of solid-ordered and liquid-ordered phase states, according to the occurrence of ripples and other higher-dimensional bilayer deformations. Evidently, the natural mixed-chain sphingomyelin does not assume the flat L beta, phase but instead the rippled P beta, phase, with symmetric and asymmetric ripples as well as macroripples and an egg-carton pattern, depending on the incubation conditions. An unexpected difference was observed between samples that are hydrated above and below the phase transition temperature. When the lipid is hydrated at low temperature, a sponge-like network of bilayers is formed in the gel state, next to some normal lamellae. The network loses its ripples during cold-incubation, which indicates the formation of a liquid-ordered (lo) gel phase. Ripples re-appear upon warming and the sponge-like network disintegrates spontaneously and irreversibly into small vesicles above the phase transition.     

Acid-induced fusion of liposomes: studies with 2,3-seco-5 alpha-cholestan-2,3-dioic acid

The effect of 2,3-seco-5 alpha-cholestan-2,3-dioic acid on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine is markedly dependent on pH. Above pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid raises the temperature of this transition, i.e., it stabilizes the bilayer phase. At pH 6.56 there is little effect of this sterol derivative on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. However, below pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid markedly lowers the temperature of this transition. The promotion of hexagonal phase formation increases both with increasing mol fraction of this sterol derivative and with lower pH, particularly in the range between pH 6.56 and pH 5.0. Below about pH 6, 2,3-seco-5 alpha-cholestan-2,3-dioic acid also induces vesicle fusion as measured both by lipid mixing as well as by mixing of aqueous contents. For these assays vesicles made of phosphatidylethanolamine (made from egg phosphatidylcholine) and extruded through 0.2 micron pore membranes were used. At higher concentrations or at lower pH the 2,3-seco-5 alpha-cholestan-2,3-dioic acid induces some leakage of the contents of these vesicles. Nevertheless, with vesicles containing only 2 weight% sterol derivative, it was possible to demonstrate substantial mixing of aqueous contents of the vesicles over the pH range 3.5 to 5.5. Several of the properties of 2,3-seco-5 alpha-cholestan-2,3-dioic acid indicate that this compound may be useful in sensitizing vesicles to acid-induced fusion for the purpose of endocytic drug delivery.     

Effect of temperature and pH onCandida blankii in chemostat culture

The growth characteristics ofCandida blankii as a function of temperature and pH in a simulated bagasse hemicellulose hydrolysate were determined in chemostat culture. The highest maximum specific growth rate of 0.44h^–1 was reached at 38°C and at pH 5.5, with a sharp decrease in growth rate on either side of this temperature. Growth occurred at 46°C but not at 48°C. The protein and cell yields varied little below 40°C and the respective values were 0.22 and 0.5 g/g at 38°C. At the lower pH values, a severe linear decrease in cell and protein yields occurred, whereas a small increase in these yields at decreasing pH values was found when acetic acid was omitted from the medium. In the presence of acetic acid, a very sharp decrease in the growth rate at pH values below pH 4.5 was noted, despite the very low residual acetic acid concentrations, of less than 50 mg/l, in the culture.     

Characteristics of type IV collagen unfolding under various pH conditions as a model of pathological disorder in tissue

The overall structure of type IV collagen is the same at neutral and acidic pH, as determined by circular dichroism spectra. The heating rate dependence of denaturation midpoint temperature (T(m)) shows that type IV collagen is unstable at body temperature, similarly to type I collagen. The heating rate dependence of T(m) at neutral pH has two phases, but that at acidic pH apparently has a single phase. The T(m) of the first phase (lower T(m)) at neutral pH is consistent with that at acidic pH, and the activation energy of these phases is consistent, within experimental error. The triple helix region of type IV collagen corresponding to the second phase (higher T(m)) at neutral pH is thermally stable when compared to the triple helical structure at acidic pH. At acidic pH, as the loosely packed and unstable region has spread throughout the whole molecule, the thermal transition is thought to be cooperative and is observed as a single phase. Structural flexibility is related to protein function and assembly; therefore, the unstable structure and increased flexibility of type IV collagen induced at acidic pH may affect diseases accompanied by type IV collagen disorder.     

The effect of pH on the phase transition temperature of dipalmitoylphosphatidylcholine-palmitic acid liposomes

The shift in the gel-liquid crystal phase transition temperature (tm) of dipalmitoylphosphatidylcholine liposomes induced by incorporation of 10 mol% palmitic acid, was measured by 90 degrees light scattering at different bulk pH values. It has been found that the tm shift decreases sigmoidally from 4.7 to -0.3 degrees C as the bulk pH is raised from 5 to 11. Since it is in this range that the carboxyl group of a membrane-bound fatty acid should ionize, our results can be interpreted to mean that there is relationship between the tm shift and the degree of dissociation of palmitic acid, the uncharged fatty acid increasing tm and its conjugate, anionic form, slightly decreasing the transition temperature of dipalmitoylphosphatidylcholine liposomes. The experimental results are fitted by a modified form of the Henderson-Hasselbach equilibrium expression which takes into account the effect of the anionic fatty acid on the surface potential and hence, on the surface pH of liposomes, according to Gouy-Chapman and Boltzmann equations, respectively. Best fit between theory and experiments is found when the intrinsic interfacial pK of palmitic acid is set equal to 7.7. This high pK value can be explained as due to the effect of the lower dielectric constant of the interfacial region, as compared to bulk water, on the acid-base dissociation of the carboxyl group. The results presented here show that upon incorporation of palmitic acid, the phase transition of dipalmitoylphosphatidylcholine bilayers becomes extremely sensitive to changes of pH in the vicinity of the physiological range. This property is not shown by the pure phospholipid bilayers in the same pH range.     

Recombination of S-peptide with S-protein during folding of ribonuclease S. II. Kinetic characterization of a stable folding intermediate shown by S-protein at pH 1.7.

At pH 1.7 S-peptide dissociates from S-protein but S-protein remains partly folded below 30 °C. A folded form of S-protein, labeled I₃, is detected and measured by its ability to combine rapidly with S-peptide at pH 6.8 and then to form native ribonuclease S. The second-order combination reaction (k = 0.7 × 10⁶m^?1s^?1 at 20 °C) can be monitored either by tyrosine absorbance or fluorescence emission; the subsequent first-order folding reaction (half-time, 68 ms; 20 °C) is monitored by 2′CMP ² binding. Combination with S-peptide and folding to form native RNAase S is considerably slower for both classes of unfolded S-protein (see preceding paper).I₃ shows a thermal folding transition at pH 1.7: it is completely unfolded above 32 °C and reaches a limiting low-temperature value of 65% below 10 °C. The 35% S-protein remaining at 10 °C is unfolded as judged by its refolding behavior in forming native RNAase S at pH 6.8. The folding transition of S-protein at pH 1.7 is a broad, multi-state transition. This is shown both by the large fraction of unfolded S-protein remaining at low temperatures and by the large differences between the folding transition curves monitored by I₃ and by tyrosine absorbance.The fact that S-protein remains partly folded after dissociation of S-peptide at pH 1.7 but not at pH 6.8 may be explained by two earlier observations. (1) Native RNAase A is stable in the temperature range of the S-protein folding transition at pH 1.7, and (2) the binding constant of S-protein for S-peptide falls steadily as the pH is lowered, by more than four orders of magnitude between pH 8.3 and pH 2.7, at 0 °C. The following explanation is suggested for why folding intermediates are observed easily in the transition of S-protein but not of RNAase A. The S-protein transition is shifted to lower temperatures, where folding intermediates should be more stable: consequently, intermediates in the folding of RNAase A which do not involve the S-peptide moiety and which are populated to almost detectable levels can be observed at the lower temperatures of the S-protein transition.     

Use of salicylic acid to measure the apparent intracellular pH in the ehrlich ascites-tumor cell and Escherichia coli

The distribution of salicylic acid between the intracellular and extracellular phases has been used to estimate the intracellular pH in the Ehrlich cell and Escherichia coli. The validity of the method was established by: (i) comparison of the results obtained with salicylic acid with those obtained with 5,5-dimethyloxazolidine-2,4-dione; (ii) by following changes of the apparent intracellular pH under circumstances in which such changes are predictable, e.g., the addition of weak acids or proton conductors to the incubation medium during incubation at acidic pH; (iii) by comparison of the apparent intracellular pH changes with the uptake of H⁺ by the cells estimated from the changes of the medium pH. Optimal results are obtained with this indicator when the extracellular pH is below 5.5, because in this case the indicator is to a sufficient extent in its penetrating form, so that its movement can reflect intracellular pH changes occurring in less than 30 s. When the intracellular pH falls below 5.2 measurable binding of salicylic acid to the intracellular material of the Ehrlich cell takes place, but above this pH no binding has been found.The Ehrlich cell and cells of Escherichia coli behaved similarly under various experimental circumstances tested, but striking differences were found in the inherent permeability of the membrane to H⁺ and in the changes in this parameter by lowering the temperature to 2°C.     

Ethanol production from glucose byClostridium thermosaccharolyticum strains: Effect of pH and temperature

Summary The fermentation of glucose byClostridium thermosaccharolyticum strains IMG 2811^T, 6544 and 6564 was studied in batch culture in a complex medium at different temperatures in defined and free-floating pH conditions. All the strains ferment 5 g glucose.l^–1 completely. The yield of the fermentation products turned out to be independent of the incubation temperature for strain IMG 2811^T. Strain IMG 6544 produced at 60°C significantly more ethanol and less acetic acid, butyric acid, hydrogen gas and biomass than at lower temperatures. With strain IMG 6564, the opposite effect occurred: ethanol appeared to be the main fermentation product at 45°C; at 60°C less ethanol and more acetic acid, butyric acid and hydrogen gas was formed.Experiments, carried out with strain IMG 6564, at defined pH conditions (between 5.5 and 7) and different temperatures (45, 55 and 60°C) revealed no effect of the incubation temperature, but an important effect of the pH on the product formation. At pH 7, ethanol was the main fermentation product while minor amounts of hydrogen gas, acetic and butyric acid were produced. Lowering the pH gradually to 5.5 resulted in a decrease of ethanol and an increase of biomass, hydrogen gas, acetic, butyric and lactic acids. At pH higher than 7 no growth occurred. Similar conclusions could be drawn for strains IMG 2811^T and 6544.     

Low pH induces an interdigitated gel to bilayer gel phase transition in dihexadecylphosphatidylcholine membrane.

We have investigated the influence of pH on the structures and phase behaviors of multilamellar vesicles of the ether-linked dihexadecylphosphatidylcholine (DHPC-MLV). This phospholipid is known to be in the interdigitated gel (L(beta)I) phase in excess water at 20 degrees C at neutral pH. The results of X-ray diffraction experiments indicate that a phase transition from L(beta)I phase to the bilayer gel phase occurred in DHPC-MLV in 0.5 M KCl around pH 3.9 with a decrease in pH, and that at low pH values, less than pH 2.2, DHPC-MLVs were in L(beta') phase. The results of fluorescence and light scattering method indicate that the gel to liquid-crystalline phase transition temperature (T(m)) of DHPC-MLV increased with a decrease in pH. On the basis of a thermodynamic analysis, we conclude that the main mechanism of the low-pH induced L(beta)I to bilayer gel phase transition in DHPC-MLV and the increase in its T(m) is connected with the decrease in the repulsive interaction between the headgroups of these phospholipids. As pH decreases, the phosphate groups of the headgroups begin to be protonated, and as a result, the apparent positive surface charges appear. However, surface dipoles decrease and the interaction free energy of the hydrophilic segments with water increases. The latter effect dominates the pure electrostatic repulsion between the charged headgroups, and thereby, the total repulsive interaction in the interface decreases.     

电子自旋共振波谱技术研究不同pH值对二棕榈酰磷脂酸脂质体相变性质的影响

本文用电子自旋共振(ESR)波谱技术研究了不同pH值对酸性磷脂二棕榈酰磷脂酸(DPPA)脂质体相变性质的影响。结果表明:DPPA脂质体随pH值的增加其相变温度降低。通过对测试条件的选择和波谱参数的测量,TEMPO标记的DPPA脂质体在HEPES溶液中ESR谱不出现疏水峰的原因,是由于仪器分辨率不够以及自旋标记探针TEMPO在DPPA脂质体脂双层疏水相中的超精细偶合常数Ao和各向同性go因子与它们在HEPES溶液中的值相近。     

Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate

The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to transesterified egg phosphatidylethanolamine (TPE) raised the lamellar-hexagonal transition temperature of TPE by 11 degrees C. Stable bilayer vesicles were formed when the incorporated CHEMS exceeded 20 mol %. (2) At a pH below 5.5, the protonation of CHEMS enhanced the formation of the hexagonal phase (HII) of TPE. At 25 mol % CHEMS the bilayer-hexagonal transition temperature was lowered by 30 degrees C at pH 4.5. (3) The endothermic acid-induced hexagonal hexagonal transition of TPE-CHEMS was suppressed at 35 mol % CHEMS. However, 31P NMR and electron microscopy indicated that a lamellar-hexagonal transition still occurred at this composition. (4) The main transition of TPE was not affected by the protonation of the incorporated CHEMS, indicating that no macroscopic phase separation occurred in TPE-CHEMS mixtures at low pH. (5) In contrast to the HII-promoting effect of H+, the neutralization of the negative charge on TPE-CHEMS by Ca2+ resulted in aggregates that remained in the lamellar structure even at the hexagonal transition temperature of TPE. It is suggested that calcium might form a complex between CHEMS in apposed bilayers. These results are related to the possible biological function of acidic cholesterol esters in biomembranes.