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PubMed2001

Determination of electrokinetic charge with a particle-charge detector, and its relationship to the total charge.

The main features of polyelectrolyte titrations with end-point indication by means of a particle-charge detector (PCD) were investigated. Because of their well-defined character, the charges of different synthetic polyelectrolytes and of latex samples with different functional groups were measured at different pH and ionic strength. The results show that PCD is a valuable tool for detecting effective or dissociated counterion charge without additional model assumptions. For negatively charged samples with exclusively strong acid functional groups, an excellent agreement was obtained between cation-exchange capacity and the charge measured by PCD over a wide pH range. For samples with additional carboxyl groups, the PCD charge was significantly lower than the total charge calculated from cation-exchange results. It can be concluded that counterion immobilization by a Stern layer-type arrangement is responsible for this effect.

Journal Article↗
PubMed1978

Enzyme behaviour and molecular environment. The effects of ionic strength, detergents, linear polyanions and phospholipids on the pH profile of soluble cytochrome oxidase.

The activity vs. pH profile for the oxidation of ferrocytochrome c by purified cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was investigated as a function of ionic strength (from 10 to 200 mM) in the absence and in the presence of various perturbants: Tween 20, linear polyanions (RNA, heparin, polyglutamic acid) and phospholipids (asolectin, phosphatidylcholine, phosphatidic acid and cardiolipin). The activation induced by Tween 20 and "zero net charge" phospholipid liposomes was not pH dependent. On the other hand, linear polyanions and polyanionic liposomes strongly perturbed the pH profile, mostly at low ionic strength, by shifting the pH optimum about 1.7 pH units towards alkaline pH values. This effect was reversed by increasing ionic strength. These observations are interpreted in the light of polyelectrolyte theory. Since these results show striking with membrane-bound enzyme, it is concluded that in vivo cytochrome oxidase is located within polyanionic sites of the micochondrial membrane. The activation broght about by phospholipids may result from two posible processes: creation of a hydrophobic environment by the non-polar tails, preventing autoaggregation; and creation of a suitable polyelectrolytic environment by the polar heads (of non zero net charge), increasing the intrinsic reaction rate.

Animals↗
PubMed1989

Electron transfer between liposomal cytochrome c1 and cytochrome c: catalytic implications of electrostatic potentials.

Kinetics measurements of the electron transfer between ferricytochrome c and liposomal ferrocytochrome c1 (with and without the hinge protein) were performed. The observed rate constants(kobs) of electron transfer between liposomal ferrocytochrome c1 and ferricytochrome c at different ionic strengths were measured in cacodylate buffer, pH 7.4, at 2 C. The effect of ionic strength on the rate constant(kobs) of electron transfer between liposomal cytochrome c1 and cytochrome c is far greater than that in the solution kinetics (Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108). The result demonstrates that the membrane bound cytochrome c1 creates a polyelectrolytic microenvironment which appears to be involved in the control of electron transfer and can be modulated by the ionic strength. The involvement of electrostatic potentials in the electron transfer between the membrane bound cytochrome c1 and cytochrome c is discussed in accord with the experimental results and a polyelectrolyte theory.

Cytochrome c Group↗
PubMed1984

Mechanism and dynamics of conformational ordering in xanthan polysaccharide.

The thermally induced order-disorder transition of xanthan (extracellular bacterial polysaccharide from Xanthomonas campestris) has been investigated by optical rotation, differential scanning calorimetry, stopped-flow reaction kinetics and low-angle laser light scattering, and the results have been analysed in terms of Zimm -Bragg helix-coil transition theory. The reciprocal of the transition midpoint temperature (Tm) varies linearly with the logarithm of cation (K+) the salt dependence of Tm, is in agreement with Manning polyelectrolyte theory the ordered structure. The associated increase in cation binding, calculated from the salt dependence of tm, is in agreement with the Manning polyelectrolyte theory for one of the candidate structures from X-ray diffraction, a 5(1) single helix stabilized by packing of side-chains along the polymer backbone, but not for the alternative double-helix structure that has also been proposed. At each salt concentration, the two fundamental parameters of the Zimm -Bragg theory, s and sigma, were calculated. The equilibrium constant for growth of the ordered structure (s) is derived directly from calorimetric measurement of transition enthalpy (delta Hcal ), and sigma, which quantifies the relative instability of the helix nucleus, is derived from the ratio of delta Hcal to the apparent transition enthalpy (delta Happ ) obtained by van't Hoff analysis of the optical rotation data. The temperature course of conformational ordering calculated theoretically is in good quantitative agreement with experimental results from both optical rotation and scanning calorimetry. The calculated average length of stable, ordered chain-sequences increases with decreasing temperature, but equals or exceeds the total chain length from light scattering only at temperatures more than approximately equal to 70 K below Tm, suggesting that ordered and disordered regions may co-exist within the same xanthan molecule. Consistent with this interpretation, the observed rate of conformational ordering increases sharply under conditions where the starting solution for dynamic measurements is partially ordered, suggesting that ordered sequences within each chain may act as helix nuclei for adjacent disordered regions, so that helix growth, rather than the slower nucleation process, becomes rate limiting.

Calorimetry, Differential Scanning↗
PubMed1992

Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA. Possible thermodynamic origins of the "glutamate effect" on protein-DNA interactions.

The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor (lac R) protein with the strong-binding symmetric lac operator (Osym) DNA site. To this end, binding constants Kobs and their power dependences on univalent salt (MX) concentration (SKobs = d log Kobs/d log[MX]) have been determined for the interactions of lac R with Osym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, Kobs at fixed salt concentration [KX] increases when chloride (Cl-) is replaced by the physiological anion glutamate (Glu-). At 0.25 M-KX, the increase in Kobs for Osym is observed to be approximately 40-fold, whereas for non-operator DNA the increase in Kobs is estimated by extrapolation to be approximately 300-fold. For non-operator DNA, SKobsRD is independent of salt concentration within experimental uncertainty, and is similar in KCl (SKobs,RDKCl = -9.8(+/- 1.0) between 0.13 M and 0.18 M-KCl) and KGlu (SKobs,RDKGlu = -9.3(+/- 0.7) between 0.23 M and 0.36 M-KGlu). For Osym DNA, SKobsRO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SKobsRO are less than SKobsRD, and, for specific binding decrease in the order [SKobsRO,KCl[>[SKobsRO,KAc[>[SKobsRO,KGlu[ . Neither KobsRO nor SKobsRO is affected by the choice of univalent cation M+ (Na+, K+, NH4+, or mixtures thereof, all as the chloride salt), and SKobsRO is independent of [MCl] in the range examined (0.125 to 0.3 M). This behavior of SKobsRO is consistent with that expected for a binding process with a large contribution from the polyelectrolyte effect. However, the lack of an effect of the nature of the cation on the magnitude of KobsRO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH4+ > K+ > Na+) observed by 23Na nuclear magnetic resonance. For both specific and non-specific binding, the large stoichiometry of cation release from the DNA polyelectrolyte is the dominant contribution to SKobs. To interpret these data, we propose that Glu- is an inert anion, whereas Ac- and Cl- compete with DNA phosphate groups in binding to lac repressor. A thermodynamic estimate of the minimum stoichiometry of water release from lac repressor and Osym operator (210(+/- 30) H2O) is determined from analysis of the apparently significant reduction in [SKobsRO,KGlu[ with increasing [KGlu] in the range 0.25 to 0.9 M. According to this analysis, SKobs values of specific and non-specific binding in KGlu differ primarily because of the release of water in specific binding. In KAc and KCl, we deduce that anion competition affects Kobs and SKobs to an extent which differs for different anions and for the different binding modes.

Anions↗
PubMed1996

Characterization and solution properties of a new exopolysaccharide excreted by the bacterium Alteromonas sp. strain 1644.

This paper concerns the study of a new bacterial exopolysaccharide. The ionic properties of the polymer are considered in relation to the first structural results. This polysaccharide is a carboxylic polyelectrolyte with a charge parameter of 1.33, and exhibits usual polyelectrolyte properties with monovalent and Mg2+ ions. In contrast, its affinity for the divalent ions Ca2+, Sr2+ and Ba2+ is greater than predicted from electrostatic theory. In solution, the polysaccharide undergoes a local conformational transition related to the number of divalent ions bound per carboxylic group. This interaction is mainly an intramolecular process. The dimensions of the polymeric chain were also considered using light-scattering and viscometric measurements. The intrinsic persistence length Lp is equal to 80 +/- 5 A and allows this polymer to be classified as a semi-rigid chain.

Calcium↗
PubMed1995

Microcapsules through polymer complexation. Part 3: Encapsulation and culture of human Burkitt lymphoma cells in vitro.

Methacrylic acid (MAA) based polyelectrolytes were complexed with protonated or quaternized dimethylaminoethyl methacrylate (DMAEMA) containing polyelectrolytes to form microcapsules in vitro. Anchorage independent human Burkitt lymphoma (Raji) cells were successfully cultured in the presence of dissolved MAA containing polymer. Capsule morphology was investigated by light microscopy and by scanning electron microscopy (SEM). Capsules based on quaternized DMAEMA containing polymer were found to be more stable than capsules containing protonated DMAEMA functionality. Raji cells were successfully encapsulated in both systems and divided to confluence; thereafter sufficient pressure was exerted to burst open the capsules. Cells released from these capsules appeared to suffer no discernible trauma and were successfully isolated and subcultured to confluence.

Burkitt Lymphoma↗
PubMed1982

Localization of polyphosphates in Saccharomyces fragilis, as revealed by 4',6-diamidino-2-phenylindole fluorescence.

The fluorescent dye 4',6-diamidino-2-phenylindole has its emission maximum at 456 nm. Fluorescence intensity at this wavelength is significantly increased by various negatively-charged polyelectrolytes. Among several polyelectrolytes tested, polyphosphates appeared to be unique in the sense that they shifted the emission maximum from 456 to 526 nm. Addition of Saccharomyces fragilis cells to a diamidinophenylindole solution caused an immediate shift of the emission maximum to 526 nm, followed by a gradual increase of fluorescence at 456 nm. The 526 nm, but not the 456 nm fluorescence was instantly quenched by non-penetrating cations, like UO2+(2). These results suggest a momentary interaction of diamidinophenylindole with polyphosphate, localized outside the plasma membrane, followed by a slow penetration of the dye into the cells, yielding increased fluorescence at 456 nm by interaction of the dye with e.g., nucleic acids. This was confirmed by fluorescence microscopy. After addition of diamidinophenylindole the yeast cells exhibited an immediate green-yellow fluorescence of the membrane, that was suppressed by UO2+(2). After longer incubation times the cytoplasm and nucleus developed a blue fluorescence.

Cell Membrane↗
PubMed1981

The release of monovalent counterions by addition of divalent counterions in coulombic interaction system of polyions (II).

In this work, the increase in activity. Deltaa+ of a monovalent counterion (Na+) resulting from the addition or a divalent ion (Cu2+) was experimentally investigated in sulfate or sulfonate polyelectrolyte solutions. It was found that in the case of polyvinylsulfonate, a+ was expressed by Deltaa+ = Delta(C++ -a++). where C(++) and a(++) represent the molar concentration and activity of added Cu2+ respectively, while in the case of dextran sulfate, the expression or Deltaa+ became Deltaa+ =2Delta(C++-a++). Considering that interactions between these cations and sulfate or sulfonale groups are Durely electrostatic, these results were analyzed in relation to the conformation of the polyelectrolytes.

Journal Article↗
PubMed1982

Electric properties and structure of DNA-restriction fragments from measurements of the electric dichroism.

The electric dichroism of 17 homogeneous DNA fragments, ranging in size from 43 to 4362 base-pairs, has been analyzed in high electric fields. The orientation of the small fragments can be described in terms of an induced dipole moment, whereas the large fragments are oriented according to a constant dipole mechanism. In the intermediate size range, DNA orients according to an induced dipole mechanism at low field strengths and according to a constant dipole mechanism at high field strengths. From these observations we propose an orientation mechanism with a saturating induced dipole. The induced dipole observed at low field strengths is saturated at a field strength Eo within a transition range Em to give a constant dipole moment at high field strengths. These parameters together with the polarizability and the limit reduced dichroism are evaluated by a least-squares analysis of the experimental data. Eo and Em are found to decrease with increasing chain length from Eo approximately 40 kV/cm (Em approximately 14 kV/cm) at 65 base-pairs to 10 kV/cm (6 kV/cm) at 194 base-pairs. The polarizability is found to increase with the square of the chain length, whereas the saturated dipole increases with chain length N at low N and goes to a limit value at high N. The temperature dependence of the orientation parameters is found to be very small. The values obtained for the limit dichroism are between -1.0 and -1.3 for chain lengths between 60 and 1000 base-pairs, whereas values around -1.4 are observed at chain lengths greater than 1000 base-pairs. These data indicate that electric fields extend the contour of DNA strands at high chain lengths from a weakly bent to a more linear form. The variations of the limit dichroism observed for short fragments suggest sequence-dependent differences in the secondary structure of the helix. The experimental results are compared with numerical calculations based on simple polyelectrolyte models. For short fragments the magnitude of several electrochemical parameters can be adequately explained by a polarization of the ion cloud around the DNA molecules. However, these polyelectrolyte models do not adequately describe the observed chain length dependence of the orientation phenomena.

Base Composition↗
PubMed1988

The theoretical distributions and diffusivities of small ions in chondroitin sulphate and hyaluronate.

The electrostatic interactions between polyionic glycosaminoglycans and small mobile ions are investigated using the Poisson-Boltzmann equation and a rod-in-cell model of the polyelectrolyte. Calculations are made for the range of polyelectrolyte concentrations and buffer compositions for which measurements of ion distributions and diffusivities are reported in a companion paper (Maroudas et al., Biophys. Chem. 32 (1988) 257). We conclude that the distribution of mobile ions is largely determined by the 'far-field' potential and is adequately described by the Poisson-Boltzmann theory and also by more approximate theories such as ideal Donnan or 'condensation' theory. The measured variations in cation diffusivities, particularly the increase in diffusivity with increasing matrix concentration at low ionic strengths, are predicted qualitatively using an approximate diffusion theory together with the calculated potential fields. However, the same theory applied to anion diffusion gives qualitatively wrong results.

Chondroitin↗
PubMed1987

Metal-binding properties of the isolated glomerular basement membrane.

The nature of binding of metal cations to the glomerular basement membrane has been investigated using isolated bovine glomerular basement membrane. Highest-affinity binding for a number of ions is attributable to the glycosaminoglycans (mostly heparan sulfate) of the membrane. Some ions, such as divalent Mn, Ca and Ni, have specific binding sites on these polymers, while for others the ion-polyelectrolyte interaction is of a non-specific nature. Both structural and binding data indicate a linear charge density of close to unity for the heparan sulfate of the glomerular basement membrane, which at the ionic composition of the plasma filtrate corresponds to a polymer surface potential of about -45 mV. Several independent observations are better explained by a model of counter-ion condensation about the glycosaminoglycans than by conventional double layer theories. These include the valence dependence of ion binding, the sharp ejection of divalent ions at a critical concentration of La3+, and the relative insensitivity of 63Ni2+ binding to NaCl concentration in the neighbourhood of physiological ionic strength. In its interactions with metal ions, the glomerular basement membrane behaves like a dilute solution of polyelectrolytes. This conclusion has important consequences for the extent of charge reduction of the filtration barrier of the kidney, bathed as it is in an electrolyte solution of mainly monovalent salts.

Animals↗
PubMed2005

A need for caution in the use of frontal analysis continuous capillary electrophoresis for the determination of ligand binding data.

Attention is drawn to a need for caution in the determination of binding data for protein-polyelectrolyte interactions by frontal analysis continuous capillary electrophoresis (FACCE). Because the method is valid only for systems involving comigration of complex(es) and slower-migrating reactant, establishing conformity with that condition is clearly a prerequisite for its application. However, that requirement has not been tested in any published studies thus far. On the basis of calculated FACCE patterns, presented to illustrate features by which such comigration of complex(es) and slower-migrating reactant can be identified, the form of the published pattern for a beta-lactoglobulin-poly(styrenesulfonate) mixture does not seem to signify the migration behavior required to justify its consideration in such terms. Additional experimental studies are therefore needed to ascertain the validity of FACCE as a means of determining binding data for the characterization of protein-polyelectrolyte interactions.

Electrophoresis, Capillary↗
PubMed2005

Charge patch attraction and reentrant condensation in DNA-liposome complexes.

We investigated the formation of complexes between cationic liposomes built up by DOTAP and three linear anionic polyions, with different charge density and flexibility, such as a single-stranded ssDNA, a double-stranded dsDNA and the polyacrylate sodium salt [NaPAA] of three different molecular weights. Our aim is to gain further insight into the formation mechanism of polyion-liposome aggregates of different sizes (lipoplexes), by comparing the behavior of DNA with a model polyelectrolyte, such as NaPAA, with approximately the same charge density but with a higher flexibility. We employed dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, in order to explore both the hydrodynamic and structural properties of the aggregates resulting from polyion-liposome interaction and to present a comprehensive picture of the complexation process. The phenomenology can be summarized in a charge ratio-dependent scenario, where the main feature is the formation of large equilibrium clusters due to the aggregation of intact polyion-coated vesicles. At increasing polyion-liposome ratio, the size of the clusters continuously increases, reaching a maximum at a well-defined value of this ratio, and then decreases ("reentrant" condensation). The aggregation mechanism and the role of the polyion charge density in the complex formation are discussed in the light of the recent theories on the correlated adsorption of polyelectrolytes at charged interfaces. Within this framework, the phenomena of charge inversion and the reentrant condensation, peaked at the isoelectric point, finds a simple explanation.

Acrylic Resins↗
PubMed2005

Antibody immobilisation on the metal and silicon surfaces. The use of self-assembled layers and specific receptors.

The use of Staphylococcal protein A and lectins as intermediate immobilising agents allows operators to orient antibodies (Ab) towards the solution due to the presence of a specific binding sites of immunoglobulin (Ig) molecules. Antibodies of different species of animals have unequal affinities to individual lectins. The effective thickness of immobilised Ab's depends on the type of substrates used and increases in the following sequence: bare gold or silicon surface, the surface treated with self-assembled polyelectrolytes (PESA) or with protein A or some lectins deposited on the preliminary formed polyelectrolyte layer. The glycolysated protein of jp51 may be selectively immobilised from the mixture of retroviral proteins (p24 and jp51), if it is necessary to distinguish infected animals from preliminarily immunised ones by means of a vaccine based on p24 protein. It was shown that the use of Staphylococcal protein A, instead of some lectins as intermediate layer for the Ab immobilisation, does not lead to a more sensitive determination of such low-weight toxins as 2,4-dichlorophenoxyacetic acid (2,4-D). The above-mentioned results were obtained with surface plasmon resonance (SPR) technique.

Antibodies↗
PubMed2005

Response of intestinal cells and macrophages to an orally administered cellulose-PEG based polymer as a potential treatment for intractable edemas.

The elimination of water from the body represents a fundamental therapeutic goal in those diseases in which oedemas occur. Aim of this work is the design of a material able to absorb large amount of water to be used, by oral administration, in those cases in which resistance to diuretics appears. Sorption and mechanical properties of the cellulose based superabsorbent hydrogel acting as a water elimination system have been modulated through the insertion of molecular spacers between the crosslinks. Starting polymers are the sodium salt of carboxymethylcellulose (CMCNa), a polyelectrolyte cellulose derivative, and the hydroxyethylcellulose (HEC), a non-polyelectrolyte derivative. Polyethyleneglycol (PEG) with various molecular weights, has been linked by its free ends at two divinylsulfone (DVS) crosslinker molecules, in order to increase the average distance between two crosslinking sites and thus acting as spacer. Both the effect of concentration and molecular weight of the spacer resulted to significantly affect the hydrogel final sorption properties and thus the efficiency of the body water elimination system. Biocompatibility studies have been performed to test the hydrogel compatibility with respect to intestinal and macrophages cell lines. To investigate the effects of intestinal cells conditioned media after the contact with the gel on macrophages nitric oxide release tests have been carried out.

Absorption↗
PubMed2005

Fabrication, characterization, and biological assessment of multilayered DNA-coatings for biomaterial purposes.

This study describes the fabrication of two types of multilayered coatings onto titanium by electrostatic self-assembly (ESA), using deoxyribosenucleic acid (DNA) as the anionic polyelectrolyte and poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Both coatings were characterized using UV-vis spectrophotometry, atomic force microscopy (AFM), X-ray photospectroscopy (XPS), contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and for the amount of DNA immobilized. The mutagenicity of the constituents of the coatings was assessed. Titanium substrates with or without multilayered DNA-coatings were used in cell culture experiments to study cell proliferation, viability, and morphology. Results of UV-vis spectrophotometry, AFM, and contact angle measurements clearly indicated the progressive build-up of the multilayered coatings. Furthermore, AFM and XPS data showed a more uniform build-up and morphology of [PDL/DNA]-coatings compared to [PAH/DNA]-coatings. DNA-immobilization into both coatings was linear, and approximated 3microg/cm(2) into each double-layer. The surface morphology of both types of multilayered DNA-coatings showed elevations in the nanoscale range. No mutagenic effects of DNA, PDL, or PAH were detected, and cell viability and morphology were not affected by the presence of either type of multilayered DNA-coating. Still, the results of the proliferation assay revealed an increased proliferation of primary rat dermal fibroblasts on both types of multilayered DNA-coatings compared to non-coated controls. The biocompatibility and functionalization of the coatings produced here, will be assessed in subsequent cell culture and animal-implantation studies.

Animals↗
PubMed2005

Use of coagulants in treatment of olive oil wastewater model solutions by induced air flotation.

Natural polyelectrolytes are suitable coagulants for the treatment of industrial and minicipal wastewaters because they are safe and have environmental benefits. Chitosan, a natural cationic polyelectrolyte, and other similar coagulants were used in the treatment of an olive oil water suspension as a model for the processing wastewater. The effect of chitosan, starch, alum and ferric chloride on the coagulation of oil droplets were determined by the jar test apparatus and turbidometric measurements. Olive oil emulsion samples were prepared by the use of surface active agents and other agents that could form stable oil water emulsions. The effect of parameters such as pH, ionic strength and optimum dosage of the coagulants were determined in the jar test experiments. Following the jar experiments, with the optimum concentration of the suitable coagulant, the emulsions were placed in an induced air flotation (IAF) cell to separate the coagulated oil droplets from solution. In the air flotation experiments, the effect of temperature, surfactant concentration and air flowrate were determined on the decrease of turbidity and COD of the emulsion samples. In the jar experiments, chitosan and alum used together at concentrations of 15 and 25 ppm, respectively, at pH 6 produced the lowest turbidity values. In the air flotation experiments, a concentration of 100 ppm of chitosan, an air flowrate of 3 l/min, aeration time of 45 s, temperature of 20 degrees C and pH 6 produced optimum levels. At optimum conditions of coagulation and flotation stages, the COD of the olive oil emulsion could be reduced by 95%.

Air↗