Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO₄, MgCl₂, CaCl₂) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na₂SO₄) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H⁺ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10^?3 elementary charge per Ų, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.     

Analysis of the near-ultraviolet absorption and circular dichroic spectra of parsley plastocyanin for the effects of pH and copper center conformation changes

The absorption and circular dichroic (CD) spectra of parsley plastocyanin (PC) were measured in order to determine the effects of changes in primary amino acid sequence on both the copper center and protein components of the PC molecule. The near-ultraviolet (uv) absorption and CD spectra of parsley PC were found to be qualitatively similar to those of spinach, poplar, and lettuce PC, except for the near-uv CD spectrum of the reduced form at low pH (ca. pH 5.0). The CD spectrum of reduced parsley PC in the 250-265 nm wavelength region changes from positive to negative ellipticity upon reduction of pH, and is characterized by a pKa value of 5.7. This pKa value is the same as that for the protonation of the histidine 87 copper ligand, observed by NMR, and the change in conformation of the copper center. Similar processes are believed to occur in the other PC species at lower pH values. Thus, the pH-dependent perturbations of the near-uv CD spectra of reduced PC are interpreted as due to transitions in the reduced copper center. The increase in the near-uv absorption spectrum of reduced PC can be divided into pH-independent and pH-dependent portions. The pH-independent portion resembles the absorption spectrum of tetrahedral Cu(I) metallothionein, suggesting the presence of Cu(I)-Cys 84 and/or Cu(I)-Met 92 charge transfer transitions in the near-uv absorption spectra of reduced PC. The pH dependence of the absorption spectrum changes and the pH difference absorption spectrum indicate that tyrosine residues may contribute to at least a part of the pH-dependent portion of the absorption increase of reduced PC.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values <6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Positive surface charge inhibition of phospholipase A2 in mixed monolayer systems

Inhibition of pancreatic phospholipase A₂ by surface-active local anesthetics was recently reported by this laboratory to be due to enzyme-anesthetic interaction in the subphase and surface effects. In order to study surface effects in the absence of subphase effects, a long-chain tetracaine analog which was completely insoluble in the subphase, dimethylaminoethyl p-decoxybenzoate, was synthesized. To determine if inhibition was due to the positive surface charge of the analog or some other effect related to structure, the analog's inhibitory effects were compared with those of octadecylamine. Analog-didecanoyl lecithin (PC) monolayers showed nonideal mixing as evidenced by a condensing effect, while octadecylamine-didecanoyl PC monolayers showed ideal mixing. The apparent pK′_a of octadecylamine-dioctanoyl PC micelles (1:4) was 9.9, while that of the analog-dioctanoyl PC micelles (1:4) was 7.6. At pH values where both amines were fully protonated, inhibition of both porcine pancreatic and Crotalus adamanteus phospholipase A₂ on the mixed films was maximal and similar (94–97%). Inhibition decreased with increasing pH and decreasing surface charge on both mixed films and at pH values where both amines were 50% protonated, inhibition was half-maximal. At pH 8.5, where the analog was unprotonated, no inhibition was observed. Thus, inhibition of phospholipase A₂ appears to be due to a positive surface charge alone rather than any effects related to anesthetic structure or spacing in the monolayer.     

Lipid dependence of surface conformations of protein kinase C

The change of conformation of protein kinase C interacting with the surface of a mercury electrode directly from a solution or through a lipid monolayer was inferred from the number of cystine residues exposed and reduced on the electrode and from their reduction potentials. Soluble protein kinase C was estimated to have 5-6 disulfide bonds which could potentially react with the mercury electrode. Two major reduction peaks of cystine at different microenvironments within the protein molecule adsorbed to a mercury surface. They were observed in a.c. polarograms and cyclic voltamograms at two distinct potentials. The potential of these peaks became more negative as the pH of the solution increased, which was consistent with relaxation or decrease in alpha-helicity (ordered structure) of the protein as determined by circular dichroism (CD) estimations of secondary structure. The peak at the more positive potentials (-0.46 V relative to NAg/AgCl electrode at pH 7.4) tended to vanish upon cyclic reduction and reoxidation of the cystine, while the more negative peak (-0.62 V at pH 7.4) was enhanced. Addition of Mg2+ or Ca2+ had no significant effect on the potential but there was a reduction in their amplitude which appeared to affect the disappearance of these peaks upon pH adjustment. This suggests that the tertiary structure of the molecule is stabilized by Ca2+ and Mg2+, as substantiated by CD spectral analysis of secondary structures. Protein kinase C penetrated lipid monolayers to some extent. Addition of diacylglycerol or phorbol ester to the lipid monolayers facilitated this penetration. These compounds stabilized the protein surface conformation by destabilizing the monolayer at more positive potentials, resulting in an enhanced reduction peak at -0.42 V. This phenomenon was not significantly affected by Mg2+ or by Ca2+. The region of the protein kinase C (PKC) sequence which penetrated the monolayer contains cysteines and a primary amine(s), and may have homology to a region of phospholipase A2 which has been proposed as a phospholipid binding site for the two enzymes. Additionally, these polarographic studies suggest that PKC associates with and penetrates monolayers in a divalent cation-independent manner in agreement with our previous physical analyses of PKC interactions with lipid bilayers.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Electrostatically induced change of the conformational order of charged lipid membranes

Raman spectroscopy and X-ray diffraction are used to investigate the influence of surface charges on the structure of ionizable lipid membranes of dimyristoylmethylphosphatidic acid. The membrane surface charge density is regulated by varying the pH of the aqueous phase. Changes of the conformational order of the lipid chains are determined from the intensity of the CC stretch chain vibrations around 1100 cm^?1 in a lipid Raman spectrum. In going from an electrical neutral to a negatively charged membrane, the conformational order is reduced by 5% in the ordered and by 9% in the fluid membrane phase, corresponding to 0.6 and 0.8 CC bonds, respectively, which change from a trans to a gauche conformation. The electrostatically induced conformational change is mainly concentrated at the lipid chain ends as indicated by the spectral variations of the 890 cm^?1 CH₃ rocking band of the chain termini. The X-ray diffraction experiments show that increasing the surface charge density in the ordered membrane phase leads to a lateral expansion of the packing of the lipid polar groups, whereas the packing of the lipid chains in a plane perpendicular to the chain axes remains constant, indicating an increase of the tilt of the lipid chains from δ = 10° (pH 3) to δ = 27° (pH 9).     

Human placental alkaline phosphatase: Effects on conformation by ligands which alter catalytic activity

The conformation of human placental alkaline phosphatase (EC 3.1.3.1) has been studied using the spectroscopic structural probes of pH difference spectroscopy, solvent perturbation difference spectroscopy, and circular dichroism. Of the 37 ± 1 tyrosine residues in placental alkaline phosphatase (PAP), 5 ± 1 residues are observed by pH difference spectroscopy to be “free” and presumed to be located on the surface of the enzyme molecule. The ionization of these 5 “free” tyrosyl groups is not time dependent and is reversible with a pK_app of 10.29. The remaining 32 ± 1 tyrosines are considered “buried” and ionization is observed to be both time dependent and irreversible. Treatment of the enzyme with 4 m guanidine-hydrochloride normalizes all 37 ± 1 tyrosine residues (pK_app = 10.08). The difference pH titration studies thus provide spectrophotometric evidence for a change in molecular conformation of PAP in the pH region of 10.5. Using solvent perturbation difference spectroscopy and circular dichroism, the local environments of tyrosine and tryptophan residues were elucidated for the native enzyme and the enzyme in the presence of ligands that influence catalytic function: inorganic phosphate (competitive inhibitor), l-phenylalanine (uncompetitive inhibitor), d-phenylalanine (noninhibitor). and Mg²⁺ ion (activator). The spectral observations from these studies led to the following interpretations: (i) the binding of inorganic phosphate, a competitive inhibitor, induces a conformational change in the enzyme that may alter the active site and thereby decrease enzyme catalytic function; (ii) perturbation with l-phenylalanine gives spectral results indicating a conformational change consistent with the postulate that this uncompetitive inhibitor prevents the dissociation of the phosphoryl enzyme intermediate; and (iii) Mg²⁺ ion causes a slight separation of the enzyme subunits, which could increase accessibility to the active site and, thus, enzyme activity.     

Intermolecular interactions of influenza M1 proteins on the model lipid membrane surface: A study using the inner field compensation method

M1 protein binding to the lipid bilayer membrane (BLM) was recorded by the inner field compensation technique as a change of the boundary potential. After the protein was added to the bulk solution, the M1 adsorption produced a slow increase in boundary potential to a stationary value that was reached within the time period dependent on the quantity of the added protein. The stationary value of the potential grew with the decrease of pH or KCl concentration in the medium and was higher in the presence of negatively charged lipids in the BLM. It was shown that the potential growth with the decrease of pH is due to an increase of M1 molecule charge and not due to the increase of the M1 surface concentration or to the change of lipid charge. As the potential did not change after the removal of the protein from the bulk solution, we consider the protein adsorption on the BLM irreversible. The obtained results suggest that the protein adsorption is influenced by both electrostatic and hydrophobic interactions of M1 molecules with each other and with lipid membrane. We offer a mechanism of dissociation of the viral shell formed by M1 matrix protein. The protein shell is destabilized due to electrostatic repulsion of protein molecules caused by the increase of their positive charge.     

Phosphatidic acid and phosphatidylserine have distinct structural and functional interactions with the nicotinic acetylcholine receptor

Bilayers containing phosphatidylcholine (PC) and the anionic lipid phosphatidic acid (PA) are particularly effective at stabilizing the nicotinic acetylcholine receptor (nAChR) in a functional conformation that undergoes agonist-induced conformational change. The physical properties of PC membranes containing PA are also substantially altered upon incorporation of the nAChR. To test whether or not the negative charge of PA is responsible for this "bi-directional coupling," the nAChR was reconstituted into membranes composed of PC with varying levels of the net negatively charged lipid phosphatidylserine (PS). In contrast to PA, increasing levels of PS in PC membranes do not stabilize an increasing proportion of nAChRs in a functional resting conformation, nor do they slow nAChR peptide hydrogen exchange kinetics. Incorporation of the nAChR had little effect on the physical properties of the PC/PS membranes, as monitored by the gel-to-liquid crystal phase transition temperatures of the bilayers. These results show that a net negative charge alone is not sufficient to account for the unique interactions that occur between the nAChR and PC/PA membranes. Incorporation of the receptor into PC/PS membranes, however, did lead to an altered head group conformation of PS possibly by recruiting divalent cations to the membrane surface. The results show that the nAChR has complex and unique interactions with both PA and PS. The interactions between the nAChR and PS may be bridged by divalent cations, such as calcium.     

Dielectric studies of aqueous solutions of poly(L-glutamic acid)

The dielectric features of poly(L -glutamic acid) are studied by the Fourier synthesized pseudorandom noise method in a time domain combined with a four-electrode cell. Polymer concentration dependence, the effect of the solvent viscosity, salt effects, and pH dependence are studied concomitantly with measurements of CD. A helix-to-coil transition occurs near pH 5.6 for a salt-free solution; at higher pH values, the polymer has an ionized random-coil conformation, and at lower pH, it has a deionized α-helical conformation. When it is in the ionized random-coil conformation, with the usual features of an electrolytic polymer, the solution shows a relaxation spectrum with a large dielectric increment at low frequencies. In the deionized α-helical state, no distinct relaxation curves are obtained, which does not deny the existence of a permanent peptide dipole. The pH dependence of the dielectric increment does not mainly correspond to the conformational change from helix to coil, but rather corresponds to the change of chain expansion on account of a charge–charge interaction under low ionic strength, which is conceived of by a viscosity measurement.     

The mechanism of reduction of cytochrome c as studied by pulse radiolysis.

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.     

An experimental test of new theoretical models for the electrokinetic properties of biological membranes. The effect of UO2++ and tetracaine on the electrophoretic mobility of bilayer membranes and human erythrocytes

For a large smooth particle with charges at the surface, the electrophoretic mobility is proportional to the zeta potential, which is related to the charge density by the Gouy-Chapman theory of the diffuse double layer. This classical model adequately describes the dependence of the electrophoretic mobility of phospholipid vesicles on charge density and salt concentration, but it is not applicable to most biological cells, for which new theoretical models have been developed. We tested these new models experimentally by measuring the effect of UO2++ on the electrophoretic mobility of model membranes and human erythrocytes in 0.15 M NaCl at pH 5. We used UO2++ for these studies because it should adsorb specifically to the bilayer surface of the erythrocyte and should not change the density of fixed charges in the glycocalyx. Our experiments demonstrate that it forms high-affinity complexes with the phosphate groups of several phospholipids in a bilayer but does not bind significantly to sialic acid residues. As observed previously, UO2++ adsorbs strongly to egg phosphatidylcholine (PC) vesicles: 0.1 mM UO2++ changes the zeta potential of PC vesicles from 0 to +40 mV. It also has a large effect on the electrophoretic mobility of vesicles formed from mixtures of PC and the negative phospholipid phosphatidylserine (PS): 0.1 mM UO2++ changes the zeta potential of PC/PS vesicles (10 mol % PS) from -13 to +37 mV. In contrast, UO2++ has only a small effect on the electrophoretic mobility of either vesicles formed from mixtures of PC and the negative ganglioside GM1 or erythrocytes: 0.1 mM UO2++ changes the apparent zeta potential of PC/GM1 vesicles (17 mol % GM1) from -11 to +5 mV and the apparent zeta potential of erythrocytes from -12 to -4 mV. The new theoretical models suggest why UO2++ has a small effect on PC/GM1 vesicles and erythrocytes. First, large groups (e.g., sugar moieties) protruding from the surface of the PC/GM1 vesicles and erythrocytes exert hydrodynamic drag. Second, charges at the surface of a particle (e.g., adsorbed UO2++) exert a smaller effect on the mobility than charges located some distance from the surface (e.g., sialic acid residues).     

Membrane surface-charge titration probed by gramicidin A channel conductance.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.     

In Situ Measurement of Solid Electrolyte Interphase Evolution on Silicon Anodes Using Atomic Force Microscopy

In situ measurements of the growth of solid electrolyte interphase (SEI) layer on silicon and the lithiation‐induced volume changes in silicon in lithium ion half‐cells are reported. Thin film amorphous silicon electrodes are fabricated in a configuration that allows unambiguous separation of the total thickness change into contribution from SEI thickness and silicon volume change. Electrodes are assembled into a custom‐designed electrochemical cell, which is integrated with an atomic force microscope. The electrodes are subjected to constant potential lithiation/delithiation at a sequence of potential values and the thickness measurements are made at each potential after equilibrium is reached. Experiments are carried out with two electrolytes—1.2 m lithium hexafluoro‐phosphate (LiPF₆) in ethylene carbonate (EC) and 1.2 m LiPF₆ in propylene carbonate (PC)—to investigate the influence of electrolyte composition on SEI evolution. It is observed that SEI formation occurs predominantly during the first lithiation and the maximum SEI thickness is ≈17 and 10 nm respectively for EC and PC electrolytes. This study also presents the measured Si expansion ratio versus equilibrium potential and charge capacity versus equilibrium potential; both relationships display hysteresis, which is explained in terms of the stress–potential coupling in silicon.     

Lipid and cell membranes in the presence of gadolinium and other ions with high affinity to lipids. 2. A dipole component of the boundary potential on membranes with different surface charge

When Gd3+, a trivalent lanthanide, binds phospholipids with a high affinity, it elicits strong electrostatic effects on the surface of the lipid bilayer. Two experimental methods were applied to monitor the changes in the boundary and surface potentials induced by Gd3+ adsorption on liposomes and planar lipid bilayer membranes (BLM) made from phosphatidylserine (PS), phosphatidylcholine (PC) and their mixtures. The membrane surface charge density was changed by either varying the PS/PC ratio or by changing the degree of PS headgroup ionization in the range of pH between 2.5 and 7.5. The Gouy-Chapman-Stern (GCS) theory combined with the condition of mass balance in the experimental cell was used for quantitative treatment of ion adsorption and related changes in the diffuse part of the electrical double layer (surface potential). Data obtained using microelectrophoresis of liposome suspensions were well described within the framework of the modified GCS theory with constants of 5.10(4) and 10(3) M-1 for Gd3+ association with PS and PC, respectively (Yu. A. Ermakov, A. Z. Averbakh, and S. I. Sukharev, Biol. Membrany 14:434-445 (1997) (in Russian)). The intramembrane field compensation (IFC) technique used to study Gd3+ adsorption on planar lipid bilayers by monitoring the entire boundary potential gave completely different results. An observed drastic difference (approximately 140 mV) between the changes of boundary and surface potential was interpreted as the change in the dipole potential induced by binding of Gd3+. The magnitude of the surface dipole increased with the concentration of PS in PS/PC mixtures and became significant at most negative surface charges (more than 80% of PS in the mixture) and strongly correlated with the degree of PS ionization at different pH. The nature of structural changes at the membrane/water interface induced by Gd(3+)-PS interaction and possible lipid clusterization are discussed in the context of their biological importance.     

Plastocyanin conformation. An analysis of its near ultraviolet absorption and circular dichroic spectra.

The near-ultraviolet absorption and circular dichroic spectra of plastocyanin are dependent upon the redox state, solution pH, and ammonium sulfate concentration. This dependency was observed in plastocyanin isolated from spinach, poplar, and lettuce. Removal of the copper atom also perturbed the near-ultraviolet spectra. Upon reduction there are increases in both extinction and ellipticity at 252 nm. Further increases at 252 nm were observed upon formation of apo plastocyanin eliminating charge transfer transitions as the cause. The spectral changes in the near-ultraviolet imply a flexible tertiary conformation for plastocyanin. There are at least two charge transfer transitions at approximately 295-340 nm. One of these transitions is sensitive to low pH's and is attributed to the His 87 copper ligand. The redox state dependent changes observed in the near-ultraviolet spectra of plastocyanin are attenuated either by decreasing the pH to 5 or by increasing the ammonium sulfate concentration to 2.7 M. This attenuation cannot be easily explained by simple charge screening. Hydrophobic interactions probably play an important role in this phenomenon. The pH and redox state dependent conformational changes may play an important role in regulating electron transport.     

Surface potential and reaction of membrane-bound electron transfer components. I. Reaction of P-700 in sonicated chloroplasts with redox reagents

Salt- or pH-induced change of the rate of reduction of the phtooxidized membrane bound electron transfer components, P-700, by ionic and nonionic reductants added in the outer medium was studied in sonicated chloroplasts.

The rate with the negatively charged reductants increased with the increase of salt concentration at a neutral pH or with the decrease of medium pH. Salts of divalent cations were much more effective than those of monovalent cations. A trivalent cation was even more effective. The rate with a nonionic reductant was little affected by salts.

The change of the reduction rate was analyzed using the Gouy-Chapman theory, which explains the change of reduction rate by the changes of activities of ionic reductants at the charged membrane surface where the reaction takes place. This analysis gave more useful parameters and explained more satisfactorily the case with high-valence cation salts than the Brönsted type analysis. The values for the surface charge density and the surface potential of the membrane surface in the vicinity of P-700 estimated from the analysis were lower than those estimated for the surface in the vicinity of Photosystem II primary acceptor, suggesting the heterogeneity of the thylakoid surface.

The salt-induced surface potential change was shown to affect the activation energy of the reaction between P-700 and the ionic reagent.