9-Aminoacridine fluorescence changes as a measure of surface charge density of the thylakoid membrane

1. When suspended in a low cation-containing medium, chloroplast thylakoid membranes and carboxymethyl-cellulose particles quench the fluorescence from 9-aminoacridine (Searle, G.F.W. and Barber, J. (1978) Biochim. Biophys. Acta 502, 309–320).2. Relief of this quenching is achieved by adding cations to the suspension medium with the order of effectiveness being C³⁺ > C²⁺ > C⁺, indicating that the fluorescence acts as an indicator of the surface electrical potential.3. Using the Gouy-Chapman theory, the differential effect of divalent (methyl viologen) and monovalent (K⁺) cations has been used to calculate surface charge densities.4. The calculations indicate that the surface charge density on the thylakoids significantly increases when cations are added to the low cation-containing medium. Under the same conditions the surface charge density of glutaralde-hyde-fixed thylakoids and carboxymethyl-cellulose particles remained essentially constant.5. It is argued that the 9-aminoacridine technique is able to probe localized areas on the membrane surface and that the variability of the surface charge density of untreated thylakoids may be due to redistribution of charges associated with membrane stacking as suggested by Barber and Chow (Barber, J. and Chow, W.S. (1979) FEBS Lett. 105, 5–10).     

Binding of 9-aminoacridine to deoxydinucleoside phosphates of defined sequence: Preferences and stereochemistry

The effect of sequence on the binding of 9-aminoacridine to DNA has been investigated by studying its interaction with deoxydinucleoside phosphates of different sequences using proton nuclear magnetic resonance. Quantitative binding information can be obtained by comparison of the proton chemical shift behavior of 9-aminoacridine upon addition of dinucleoside phosphate to various models for the interaction using least-squares computer fitting procedures. The simplest model that fits the data includes (1) dimerization of 9-aminoacridine and (2) a mixture of 1:1 and 2:1 (dinucleoside phosphate/9-aminoacridine) complexes. The computed parameters allow comparison of binding constants and stereochemistry for different sequences. The 1:1 complexes seem to involve interaction of the ring nitrogen with the backbone phosphate and stacking of one or both chromophores on the acridine; preference in binding is observed for alternating (purine-pyrimidine or pyrimidine-purine) over non-alternating (purine-purine) dinucleoside phosphates. The 2:1 complexes involve intercalation of the acridine between two complementary dinucleoside phosphate strands with weak sequence preferences in binding. The stereochemistry of intercalation differs between non-alternating purine-purine sequences and the alternating pyrimidine-purine or purine-pyrimidine sequences in having the 9-aminoacridine stacked with the purines of one strand rather than straddling the purines on opposite strands. The difference in stereochemistry could possibly be a determining factor in frameshift sequence specificity.     

Electrostatic properties of cells estimated by absorption and fluorescence spectroscopy

A colloid titration method was used to determine the surface charge of cells of a human colon adenocarcinoma cell line WiDr; 6.2±0.8×10⁸ charges per cell were found. The apparent surface charge density was calculated using the cell surface area estimated by a Coulter counter. Alternatively, the lower limit of the cell surface area was estimated by visible microscopy. The same procedure was applied for human skin fibroblasts, resulting in the value 9.4±1.1×10⁸ charges per cell. This is significantly higher (p<0.05) than that of WiDr cells, presumably because of the different size of the cells. According to the estimations using the Coulter counter, the median diameter was higher in the case of skin fibroblasts. Fluorimetric titration of the fluorescent probe U-6 was used to estimate the interfacial potential of the WiDr cells. A shift of the titration curve of the U-6 probe toward higher pH values compared to that in pure buffer solutions was found in the presence of the WiDr cells. From the displacement of the midpoints of the titration curves, the interfacial potential of the WiDr cells was found to be about−35.8 mV. Incubation of the cells at two different pH values (7.4 and 6.8) did not result in any significant modification of the electrostatic properties of the cells under the experimental conditions of the present study. Electron microscopy revealed a distinct difference in the surface morphology of the WiDr cells compared to human skin fibroblasts. Numerous microvilli present on the surface of WiDr cells indicated marked uncertainties in cell surface area estimations. This gives large uncertainties in the real surface charge densities of cells.     

The effect of surface charge density on valinomycin-K+ complex formation in model membranes

The model membrane approach was used to investigate the surface charge effect on the ion-antibiotic complexation process. Mixed monolayers of valinomycin and lipids were spread on subphases containing K⁺ or Na⁺. The surface charge density was modified by spreading ionizable valinomycin analogs on aqueous subphases of different pH or by changing the nature of the lipid (neutral, negatively charged) in the mixed film. Surface pressure and surface potential measurements demonstrated that a neutral lipid (phosphatidylcholine) or positively charged valinomycin analogs didn't enhance the antibiotic complexing capacity. However, a maximal complexation is reached for a critical lipid concentration in the valinomycin-phosphatidylserine mixed film. The role of the surface charge on the valinomycin complexing properties was examined in terms of the Gouy-Chapman theory. As a consequence of the negative charge of the lipid monolayer, the K⁺ concentration near the surface is larger than the bulk concentration, by a Boltzmann factor. A good agreement was observed between the experimental results and the theoretical predictions. Conductance measurements of asymmetric bilayers containing a neutral lipid (egg lecithin) on one side and a negatively charged lipid (phosphatidylserine) on the other, confirm the role of the surface charge. Indeed, addition of K⁺ to the neutral side of the bilayer containing valinomycin had no effect on the conductance whereas addition of K⁺ to the charged side of the bilayer caused a 80-fold conductance increase.     

Probing the effect of nitro groups in nitramine based energetic molecules: a DFT and charge density study

The structure, electron density distribution, energetic and electrostatic properties of simple nitramine based energetic TMA, DMNA, MDA and TNA molecules were determined using density functional theory (B3LYP) with the 6-311G^** and aug-cc-pVDZ basis sets coupled with Bader's theory of atoms in molecules. In the NO₂ group substituted molecules, the N–N bond distance increases with the increase of NO₂ groups, whereas in C–N bonds, this effect is relatively less, and the distances are almost equal. The topological analysis of electron density reveals that the electron density ρ_bcp(r) of C–N and N–N bonds are significantly decreasing with the increase of NO₂ groups in the nitramine molecules. The Laplacian of electron density ▽²ρ_bcp(r) of N–NO₂ bonds [DMNA: ? 16.7 eÅ^? 5, MDA: ? 12.8 eÅ^? 5 and TNA: ? 7.9 eÅ^? 5] of the molecules are relatively less negative, and the values also decrease with the increase of NO₂ groups; this implies that the charge concentration decreases with the increase of NO₂ groups, which leads to weakening the N–N bonds of the molecules. The isosurface of molecular electrostatic potential displays high electronegative regions around the NO₂ groups. The oxygen balance OB₁₀₀ of the molecules increases as the number of NO₂ group increases in the molecules, in which, the TNA molecule having maximum OB₁₀₀ value [+7.89]. The band gap, heat of detonation, bond dissociation energy and charge imbalance are predominantly depends on the number of NO₂ group present in the molecule. The charge imbalance parameter (ν) has been calculated for all molecules, which reveals that TNA is a highly sensitive molecule, the corresponding ν value is 0.047.     

ESR study of intercalation: quantitative evaluation of drug-DNA binding through competition with a spin-labeled 9-aminoacridine

Interaction of a spin-labeled 9-aminoacridine with DNA was studied by electron spin resonance spectroscopy. Accurate determination of the binding parameters, equilibrium dissociation constant (K_D), and total number of ligand-binding sites was obtained using Scatchard and Lineweaver-Burk plots. The competition between 9-aminoacridine and its spin-labeled derivative was examined by a similar analysis of the spin-label signals. The practical interest of this method lies in the fact that the precision and the simplicity of the measurements allow the quantitative determination of the binding capacity of any intercalative drug which interacts specifically with adenine/thymine bases of DNA.     

Application of 9-aminoacridine as a probe of the surface potential experienced by cation transporters in the plasma membrane of yeast cells

The applicability of 9-aminoacridine as a probe of the surface potential of yeast cells is examined. Yeast cells are found to quench the fluorescence of the dye and it is shown that this quenching is caused by a decrease in the dye concentration in the bulk aqueous phase. Consistent with predictions of the Gouy-Chapman theory the dye is displaced from the surface of the yeast cells by addition of salts, the effectiveness of the salts being related to the valency of the cation: $\text{C}{}^{\mathrm{3+}}\text{> C}{}^{\mathrm{2+}}\text{> C}{}^{\mathrm{1+}}$. It is shown that 9-aminoacridine is predominantly bound by the plasma membrane of the cells. Only a minor part of the binding occurs in the cell wall, in line with the finding that enzymic removal does not significantly affect the binding of the dye to the cells. A single relationship for the distribution ratio of the dye between cells and medium with the ζ potential of the cells is found, irrespective of the way the ζ potential is changed, either by varying the pH or the Ca²⁺ concentration. It is argued that the electrostatic potentials probed by the dye are much higher than the corresponding ζ potentials and are of the same order of magnitude of the presumed discrete charge potentials experienced by cation transporters in the plasma membrane. It is concluded that 9-aminoacridine may be applied as a convenient and almost quantitative probe of the surface potential that effects the kinetics of ion uptake by the yeast cells.     

Effect of the surface charge density of nanoparticles on their translocation across pulmonary surfactant monolayer: a molecular dynamics simulation

Interaction between nanoparticles (NPs) and pulmonary surfactant monolayer plays a very significant role in nanoparticle-based pulmonary drug delivery system. Previous researches have indicated that different properties of nanoparticles can affect their translocation across pulmonary surfactant monolayer. Here we performed coarse-grained molecular dynamics simulation aimed at nanoparticles’ surface charge density effect on their penetration behaviours. Several hydrophilic nanoparticles with different surface charge densities were modelled in the simulations. The results show that NPs’ surface charge density affects their translocation capability: the higher the surface charge densities of NPs are, the worse their translocation capability is. It will cause the structural changes of pulmonary surfactant monolayer, and inhibit the normal phase transition of the monolayer during the compression process. Besides, charged NPs can be adsorbed on the surface of the monolayer after translocation as a stable state, and the adsorption capability of NPs increases generally with the increase of surface charge densities. Our simulation results suggest that the study of nanoparticle-based pulmonary drug delivery system should consider the nanoparticles’ surface charge density effect in order to avoid biological toxicity and improve efficacy.     

Structure and function of Fc receptors : Receptors and ligands in intercellular communication: volume 2 Edited by A. Froese and F. Paraskevas Marcell Dekker; New York and Basel, 1983 294 pages. Sw.Fr. 120.00

Plasmalemma vesicles were isolated in a sucrose-containing medium from wheat and oat roots and the net negative surface charge density was determined with 9-aminoacridine fluorescence [Chow, W.S. and Barber, J. (1980) J. Biochem. Biophys. Methods 3, 173-185]. The outer surface of the vesicles (measured in the presence of sucrose) had densities of ? 16 to ? 20 mC·m^?2 and ?29 mC·m^?2 for wheat and oat roots, respectively. The inner surface - presumed to be the cytoplasmic side and calculated from the values measured in the presence and absence of sucrose - was more negative, and its size depended on the salt status of the roots.     

Interaction of antitumoral 9-aminoacridine drug with DNA and dextran sulfate studied by fluorescence and surface-enhanced Raman spectroscopy

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy are applied to study the interaction of the drug 9-aminoacridine (9AA) with DNA and dextran sulfate. The effect of the electrostatic interaction between the positively charged 9AA and negatively charged groups in relation to the excimer or exciplex emission is investigated. The exciplex emission of 9AA is connected to the intercalation of this drug between nucleic base residues. The importance of negative groups in this interaction is evaluated by using dextran and dextran sulfate as model polymers. The existence of negative charges seems to induce an increase of the drug concentration in the vicinity of the polymers. The role of electrostatic attraction in the 9AA dimerization is confirmed by the excimer emission of 9AA in the presence of dextran sulfate. In the case of DNA, the phosphate groups may induce the drug approach to the DNA chain, but the exciplex fluorescence emission could be due to a charge transfer between the drug and adenine-rich sequences of DNA.     

Probing the effect of electric field in 9,10-dimethoxy-2,6-bis(2-p-tolylethynyl)anthracene molecular nanowire using quantum chemical and charge density analysis

The effect of electric field (EF) in a newly designed molecular nanowire 9,10-dimethoxy-2,6-bis(2-p-tolylethynyl)anthracene has been analysed theoretically from the structural and electronic charge transport properties using quantum chemical and charge density calculations. The applied EF (0–0.36 VÅ^? 1) alters the molecular conformation, charge density distribution, electrostatic properties and the electronic energy levels of the molecule. Furthermore, the applied EF decreases the highest occupied molecular orbital–lowest unoccupied molecular orbital gap significantly from 1.775 to 0.258 eV and it also induces polarisation in the molecule, which leads to increase the dipole moment of the molecule. The electrostatic potential for various levels of applied EF reveals the charge-accumulated regions of the molecule. The I–V characteristics of the molecule have been studied against various applied fields using Landauer formalism.     

Surface charge on thylakoid membranes: regulation of photosynthetic electron transfer in cyanobacteria

The establishment of the steady-state rate of photosynthetic O₂ evolution by cells of Anabaena variabilis and other cyanobacteria was found to be preceded by a lag-phase the duration of which depended on the time of cell preincubation in the dark. Electron acceptors (benzoquinone, N,N,N,N-tetramethyl-p-phenylenediamine, 2,3,5,6-tetramethyl-p-phenylenediamine or 2,6-dichlorophenolindophenol) abolished the lag-phase as well as the inhibitory effect of cyanide on electron transfer. Mono-, di-and trivalent cations added to the cell suspension markedly reduced the lag-phase. As cation concentrations were increased, acceleration and subsequent deceleration of the O₂ evolution rate were observed. The efficient concentrations of cations decreased as their valency increased. The lag-phase and the rate of photosynthetic O₂ evolution by the blue-green algae are suggested to depend on the value of the membrane surface charge governing the electrostatic interaction between unidentified membrane-bound redox components. The combination of valinomycin and nigericin as well as gramicidin D enhanced the duration of the lagphase by deenergization of thylakoid membrane.Abbreviations 9AA 9-aminoacridine - BQ benzoquinone - DAD 2,3,5,6-tetramethyl-p-phenylenediamine - DPIP 2,6-dichlorophenolindophenol - FeCy ferrycyanide - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - MES 2(N-morpholino)ethane sulphonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine - Tris tris(hydroxymethyl)aminomethane     

Effects of ionic strength,serum protein and surface charge on membrane movements and vesicle production in heated erythrocytes

Morphological changes and fragmentation of human erythrocytes heated at various rates through the spectrin inactivation temperature have been examined by cinephotomicroscopy. Most cells heated in 0.20 ionic strength buffered saline developed a wavy disturbance along the cell rim when heated. Vesicles developed from the crests of the growing waves within 0.3 s of the initiation of a wave when the heating rate was 1°C/s. At an ionic strength of 0.02, only 48% of the cells developed a wave outline. The average number of waves per cell was half that at 0.2 ionic strength. When the cell surface charge was reduced by neuraminidase treatment, only 12% of the cells fragmented. Bovine serum albumin or homologous plasma also reduced fragmentation. The dependence of the wave growth on ionic strength and surface charge was broadly consistent with theoretical predictions for the growth of a displacement instability on a low interfacial tension interface. Attention has been paid to the importance of bending energy in the development of the wave. Where wave development was suppressed, the morphological changes due to heating appeared to involve membrane internalization in the region of the cell dimple.     

Structural features of a multisubstate cardiac mitoplast anion channel: Inferences from single channel recording

Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into voltage-clamped planar lipid bilayers. A low-conductance anion channel (～40 or ～85 pS in symmetric 300 or 550 mM choline Cl, respectively), characterized by the presence of two well-defined substates, at ～25 and ～50% of the fully open level, was studied in detail. The substate behavior was consistent with a multibarrelled channel containing four functionally coupled pores. At negative (cis-trans) membrane potentials, the putative portomers appeared to gate with substantial positive cooperativity, accounting for the apparent absence of a ～75% sublevel. At positive holding potentials, allosteric protomer interactions were more complicated, and the channel complex could be modeled as a dimer of dimers. The protochannels in one dimer (“dimer A”) appeared to open independently of each other, and with a relatively high probability, while the monomers comprising the second dimer (“dimer B”) were functionally coupled, could only open if both protomers in dimer A were open, and closed as soon as one of the monomers in dimer A shut. The channels also displayed Ca²⁺- (and Mg²⁺-) sensitive rectification related to bilayer lipid surface charge. By assuming that Ca²⁺ acted solely by screening surface charge, the membrane surface potential profile was used as a “microscopic ruler” to place one mouth of the channel within 10–11 Å of the bilayer surface.     

Interactions of erythrocytes with an artificial wall: influence of the electrical surface charge

Electrical charge on any biological surface plays a crucial role in its interaction with other molecules or surfaces. Here, we study, under flow conditions, the interactions of erythrocytes with an artificial surface: a platinum microelectrode whose charge density ranges from –15 to +27 μC/cm². This artificial surface could be similar in surface charge to an endothelium or a biomaterial. In this model, interactions are measured as a transient relative increase of the electrolyte resistance obtained by impedance measurement of a microelectrode. A maximal interaction of erythrocytes with the charged surface is calculated in the 0 to +10 μC/cm² charge density range. At negative surface charge, a less efficient contact was obtained because of electrostatic repulsion forces. High positive surface charge (charge density >10 μC/cm²) does not improve the contact but induces a progressive decrease in the contact efficiency, which could be explained by a rearrangement of macromolecules on the erythrocyte surface or an effect of positive groups on the cell membrane. This work suggests that a greater surface area of contact is obtained in the 0 to +10 μC/cm² charge density range and that this is provided by more molecular bridges. Received: 23 February 1996 / Accepted: 26 April 1996     

Electrostatic surface properties of plasmalemma vesicles from oat and wheat roots. Ion binding and screening investigated by 9-aminoacridine fluorescence

Right-side-out and sealed plasmalemma vesicles were isolated from roots of spring wheat (Triticum aestivum L. cv. Drabant) and oat (Avena sativa L. cv. Brighton) by two-phase partition in a medium containing sucrose (0.25 mol l^-1). Oat root plasmalemma vesicles were discovered to contain a strongly fluorescent compound with an emission maximum at 418 nm. The surface potential of the membranes was monitored by 9-aminoacridine fluorescence and the effect of protein concentration, mannitol versus sucrose, absence of osmoticum, concentrations of salt, and titrations with chelators investigated. It is concluded that i) protein concentrations of less than 50 g ml^-1 for oat and 100 g ml^-1 for wheat plasmalemma vesicles should be used to avoid serious problems with non-linearity of response of 9-aminoacridine fluorescence, ii) mannitol can be used instead of sucrose as the osmoticum, iii) the vesicles were ruptured in the absence of osmoticum allowing us to monitor both sides of the membranes, iv) plasmalemma vesicles from oat roots are more negative than vesicles from wheat roots, and v) oat and wheat root plasmalemma vesicles are isolated with about the same amounts of bound Ca²⁺ and Mg²⁺. These bound divalent cations may not, however, reflect the in-vivo conditions since the tissues were homogenised in the presence of ethylenediaminetetraacetic acid.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - c1/2 value concentration at which half of the maximum effect is observed - Mops 3-(N-morpholino)propanesulfonic acid     

Surface-enhanced Raman and steady fluorescence study of interaction between antitumoral drug 9-aminoacridine and trypsin-like protease related to metastasis processes, guanidinobenzoatase

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) were applied to study the interaction of the antitumoral drug 9-aminoacridine (9AA) with a trypsin-like protease guanidinobenzoatase (GB) extracted from a mouse Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission was detected in the emission fluorescence spectra at certain drug and enzyme concentrations. A SERS study was accomplished on silver colloids at several excitation wavelengths in order to obtain more information about the interaction mechanism. The results derived from Raman spectroscopy indicated that 9AA in the amino monomeric form may interact with the enzyme by means of two different bonds: an ionic bond with a negatively charged amino acid and a ring stacking interaction with an aromatic residue placed in the catalytic site of GB. This interaction mechanism was responsible for a strong exciplex emission detected at a longer wavelength than the expected value of the normal fluorescence emission. Moreover, the GB concentration dependence of the interaction suggested that the drug was sensitive to the quaternary structure of the enzyme.     

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes: A fluorescence probe study

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles ($\text{out > in}$) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47°C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.     

The role of membrane surface charge in the control of photosynthetic processes and the involvement of electrostatic screening

Calculations of changes of the integrated space charge density within the diffuse layer adjacent to a negatively charged membrane surface have been made using analytical expressions derived from the full non-linear Poisson-Boltzmann equation of the Gouy-Chapman theory. This electrostatic screening parameter has been examined for mixed electrolytes of valency type $\text{Z}{}^{\mathrm{1+}}\text{Z}{}^{\mathrm{1?}}$ and $\text{Z}{}^{\mathrm{2+}}\text{Z}{}^{\mathrm{1?}}$ and concentration ranges were chosen so as to compare with experimental data obtained with thylakoid membranes. The results of the analysis are consistent with previous arguments (Barber, J., Mills, J.D. and Love, A. (1977) FEBS Letts. 74, 174–181) that this screening parameter is involved in the control of salt induced chlorophyll fluorescence and thylakoid stacking changes. Phenomenological equations suggesting the origin of the variations in the integrated space charge density for various salt conditions are presented. Overall the integrated space charge density (σ^′_x) is shown to be a more satisfactory measure of both short and long range effects associated with electrostatic screening and double layer repulsion of charged surfaces than the planar space charge density (?_x.