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.     

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.     

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.     

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.     

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.     

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).     

Cooperative lipid-protein interaction. Effect of pH and ionic strength on polymyxin binding to phosphatidic acid membranes.

The binding of polymyxin-B to charged dipalmitoyl phosphatidic acid membranes has been studied as function of the external pH and of the ionic strength of the buffer solution. The phase transition curves were obtained by measuring the fluorescence depolarization of diphenyl hexatriene incorporated into the membrane with temperature. The molecular process of polymyxin binding was elucidated: 1. At an ionic strength of I greater than or equal to 0.1 mol/l a three step phase transition curve is found. A high-temperature step corresponds to the non-bound lipid. A lowered phase transition concerns to protein-bound lipid domains. This again is splitted into two steps. An inner core of the domain is characterized by a lipid-protein complex which is stabilized through hydrophobic and electrostatic interactions between polymyxin and the charged lipid. This core is surrounded by an outer belt of only hydrophobically bound molecules. This part shows a lower phase transition temperature than the inner core. 2. The binding curves of polymyxin to phosphatidic acid membranes depend strongly on the ionic strength of the water phase. The cooperativity of the binding process increases with increasing ionic strength and reaches a constant value at I greater than 0.2 mol/l. The maximum fraction of bound lipid decreases with increasing ionic strength. 3. The pH of the water phase strongly influences the cooperative binding process. At pH 6 a loss of cooperativity is observed at low ionic strength. Increasing the ion concentration to I = 0.3 mol/l recuperates the cooperativity of the binding process. At pH 3.0 no cooperative binding is obtained even at high ionic strength.     

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.     

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.     

Structural and thermotropic properties of calcium-dimyristoylphosphatidic acid complexes at acidic and neutral pH conditions.

Two kinds of calcium-dimyristoylphosphatidic acid (DMPA) complexes at acidic and neutral pH conditions were prepared in the following ways. The complex at pH 4 was obtained by adding Ca2+ to DMPA dispersion in pure water. On the other hand, the complex at pH 7.4 was obtained by adding Ca2+ to DMPA dispersion in the presence of NaOH. The stoichiometries of Ca2+ ion to DMPA molecule are 0.5-0.67 and approximately 1 for the complexes at pH 4 and 7.4, respectively. Static x-ray diffraction shows that the hydrocarbon chains of the Ca(2+)-DMPA complex at pH 4 at 20 degrees C are more tightly packed than those of the complex at pH 7.4 at 20 degrees C. Furthermore, the complex at pH 4 at 20 degrees C gives rise to several reflections that might be related to the ordered arrangement of the Ca2+ ions. These results indicate that the structure of the complex at pH 4 is crystalline-like. In the differential scanning calorimetry (DSC) thermogram, the complex at pH 7.4 undergoes no phase transition in a temperature range between 30 and 80 degrees C. On the other hand, in the DSC thermogram for the complex at pH 4, a peak appears at 65.8 degrees C in the first heating scan. In the successive second heating scan, a transition peak appears at 63.5 degrees C. In connection with the DSC results, the structural changes associated with these phase transitions were studied with temperature-scan x-ray diffraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature change of the ripple structure in fully hydrated dimyristoylphosphatidylcholine/cholesterol multibilayers.

The ripple structure was studied as a function of temperature in fully hydrated dimyristoylphosphatidylcholine (DMPC)/cholesterol multibilayers using synchrotron x-ray small-angle diffraction and freeze-fracture electron microscopy. In the presence of cholesterol, the ripple structure appears below the pretransition temperature of pure DMPC multibilayers. In this temperature range the ripple periodicity is relatively large (25-30 nm) and rapidly decreases with increasing temperature. In this region, defined as region I, we observed coexistence of the P beta' phase and the L beta' phase. The large ripple periodicity is caused by the formation of the P beta' phase region in which cholesterol is concentrated and the L beta' phase region from which cholesterol is excluded. An increase in ripple periodicity also takes place in the narrow temperature range just below the main transition temperature. We define this temperature region as region III, where the ripple periodicity increases dramatically toward the main transition temperature. In region II, between regions I and III, the ripple periodicity decreases gradually with temperature. This behavior is quite similar to that of pure DMPC. Temperature-versus-ripple periodicity curves are parallel among pure DMPC and DMPCs with various cholesterol contents. We explain this behavior in terms of a model proposed by other workers.     

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)     

Insights into the dynamics of DMSO in phosphatidylcholine bilayers.

The solvation effects of dimethyl sulfoxide (DMSO) on the phase stability of dimyristoylphosphatidylcholine (DMPC) have been fully characterized using differential scanning calorimetry (DSC) and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of the sub-, pre-, and main transitions of DMPC were found to increase linearly with increasing mole fraction of DMSO up to mole fraction X=0.13 DMSO/H(2)O. Beyond X=0.13, the pre-transition peak started to merge with the peak representing the main transition. Simultaneously, the subtransition peak began to disappear as its transition temperature also decreased. At X=0.18, with both the subtransition and pre-transition absent, the main transition between the planar gel and the liquid-crystalline phase was observed at 30.3 degrees C. Transition enthalpy values indicated that the subgel, planar gel and rippled gel phases are most stable at X=0.11, 0.16 and 0.20 DMSO/H(2)O, respectively. This demonstrates that DMSO exerts distinct effects on each respective phase and corresponding transition. Temperature-dependent fluorescence emission scans show an increase in hydration as the system proceeds from the subgel phase all the way to the liquid-crystalline phase and correlated well with the effects of DMSO on the transition temperatures of DMPC observed in our calorimetry data. Initial observations for the sub- and main transition are further confirmed by fluorescence anisotropy using DPH as a probe. The results illustrate the differences in the microviscosity of each phase and how DMSO affects the phase transitions. Ultimately, our results suggest the most likely mechanism governing the biological actions of DMSO may involve the regulation of the solvation effects of water on the phospholipid bilayer.     

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.     

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.     

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.     

Molten globule-like state of human serum albumin at low pH.

Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 M guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.     

Temperature and pH dependent changes of immunoglobulin G structure.

1. The temperature and pH functions of the myeloma IgG(K) conformation were studied by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and difference adiabatic scanning microcalorimetry. 2. The IgG studied was found to be capable of a fully reversible structural change between pH 6.5 and 6.0. A transition occurring at low pH is accompanied by an increase of exposure of the chromophores to the solvent. 3. The "alkaline state" was found to be capable of a fully reversible S-like transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of 14-15 tyrosine residues and probably by a small increase in the helicity of the protein. These changes are not accompanied by an appreciable heat effect. The thermal denaturation of the "alkaline state" occurs only at 64 degrees C in the narrow temperature interval (3-4 degrees C). 4. The "acid state" is not accompanied by S-like transition at 25-35 degrees C. The thermal denaturation of the "acid state" occurs at 54 degrees C in the wide temperature interval (8-9 degrees C). 5. It was proposed that the ionisation of the invariant histidine residues situated in the "cavity" between the constant and variable domains causes the pH transition studied. The temperature changes in the interval 25-35 degrees C are explained by the alteration of the domains interposition. Similar alterations were investigated as a result of antigen-antibody reaction.