Sol–Gel transition of alginate solution by the addition of various divalent cations: A rheological study

Several rheological properties of aqueous alginate solution have been investigated in the absence and presence of four divalent cations: Ca, Cu, Mn, and Co. The concentration dependence of viscosity in alginate solution without addition of divalent cations shows a similar behavior to that found for other polysaccharides and synthetic polymers. We have found that for Ca(II) -added systems, sol-gel transition curves for different alginate concentrations can be superimposed over the concentration range of this study by using a normalized parameter. Shear-thinning behavior has been observed for all the alginate solutions by addition of four divalent cations over sol-gel transition process. The values of exponent b in the power-law relation $ {\eta \propto \dot \gamma }$ were found in the range of ?0.3 ～ ?0.6. It is suggested that a considerable portion of divalent cations remains in a cross-linking state even under shear flow. The results obtained from thixotropy measurements indicate the existence of a major framework formed in the gel-like and well-gelled samples, which can be maintained until shear rate (or shear stress) reaches a threshold value. © 1994 John Wiley & Sons, Inc.     

A small-angle x-ray scattering study of alginate solution and its Sol–Gel transition by addition of divalent cations

The small-angle x-ray scattering (SAXS) technique has been applied to investigate solution and gel structures of alginate in the absence and presence of two divalent cations: Ca(II) and Cu(II). We have observed a broad maximum in the scattering curve, a characteristic of polyelectrolyte, for the purified alginate sample. The scattering maximum disappears in excess of added simple salt and shifts toward the higher angle region with increasing alginate concentration. Concentration dependence of the position and intensity of the maximum follows power law relations with exponents close to those predicted by theory. Data analysis shows an increase in correlation length ξ and cross-sectional diameter d₀, of polymer chains upon gelation and suggests that a dimeric structure is adopted in the junction zone, consistent with the “egg-box” model previously proposed. In the Ca(II)–alginate system, the molecular parameters ξ and d₀ are found to have good correlation with the macroscopic properties of gelation, such as gel point determined by viscosity measurements. However, for the Cu(II)–alginate system there is no clearly transitional behavior observed in ξ and d₀, implying that the junction zone may be replaced by a more uniformly distributed site binding of Cu(II) ions to the carboxyl groups of both mannuronate and guluronate residues, in confirmation of previous ¹³C-nmr results. © 1995 John Wiley & Sons, Inc.     

Critical exponents for sol–gel transition in aqueous alginate solutions induced by cupric cations

The sol–gel transition in aqueous alginate solutions of four alginate samples having different molecular weights (M_W) and M/G ratios induced by cupric cations was monitored by rheology measurements. The gel point f_gel and the relaxation critical exponent n were determined using the Winter’s criterion over the alginate concentration C_Alg of 1–4 wt%. The scaling for the zero shear viscosity η₀ before the gel point and the equilibrium modulus G_e after the gel point was established against the relative distance ε from the gel point at the concentration of C_Alg = 1 wt%, giving the critical exponents k and z. The results indicated that f_gel was almost independent of the alginate concentration and became higher for the sample with lower molecular weight. The critical exponent n decreased with the increase in C_Alg for these four Cu-alginate samples and the fractal dimension d_f estimated from n suggested a denser structure in the critical gel with high G content. The critical exponent n evaluated from k and z agreed well with n determined from the Winter’s criterion.     

13C-NMR study of the interaction of bacterial alginate with bivalent cations

The effect of bivalent cations on solutions of extracellular polymeric substances (EPS) isolated from Pseudomonas aeruginosa was monitored by means of solid-state nuclear magnetic resonance. In particular, the binding of Ca2+ and Mg2+ to the alginate in aqueous solution was studied by determining the spin-lattice relaxation rates, line widths and line shapes of 13C nuclei under variation of the ion concentration. Both cations differ strongly in their affinity towards bacterial alginate. Spectral data indicate that the strong binding capacity of calcium is connected to the formation of a chelate complex, in which binding occurs particularly with the monomer units in alternating mannuronate-guluronate blocks. In contrast to this, binding of magnesium ions was found to be much weaker and non-specific.     

13C-nmr spectroscopic investigation of two yeast cell wall β-D-glucans

By means of alkaline extraction of the cell walsl of the yeasts Saccharomyces cerevisiae and Canadida albicans, water-insoluble glucans were obtained. Methylation analysis and ¹³C-nmr investigation in dimethyl sulfoxide solution revealed the similar chemical structure of these glucans, being composed of β(1 → 3) glycosidically linked D -glucopyranosyl units with a small amount of β(1 → 6) linkages. More detailed study in dilute alkaline solutions and in the gel state at neutral pH, however, showed that an ordered helical conformation of the glucan chain is less stable in the case of the S. cerevisiae glucan in comparison with that of C. albicans. Measurements of the shift of the absorption maximum of the glucan complexes with Congo Red also demonstrated such difference. The S. cerevisiae glucan was also inable to form a gel at neutral pH. The difference in stability of helical conformation of the glucans is explained on the basis of the methylation analysis, so that the S. cerevisiae glucan possesses longer side chains, which hinder its adoption of a stable helical conformation.     

Sol–sol structural transition of aqueous agarose systems

A structural transition is reported to occur in aqueous sols of agarose, an electrically uncharged biostructural polysaccharide. The transition has no measurable effect on size dispersity on the shape of the solute polysaccharide as observed by precision photon correlation spectroscopy. It originates a low-angle pattern of scattered light similar to that which monitors phase separations in polymer blends. Thus, it must be due to some extent to spatially modulated polymer clustering, typical of spinodal decomposition. In the interval of temperatures studied, it precedes very distinctly in time the thermoreversible sol–gel transition, which is known to be promoted at higher concentrations. It also anticipates to an appreciable extent the spatial density modulation observed in the gel. Although reported here for the first time, a spinodal decomposition of the sol that precedes and possibly triggers the processes leading to gelation does not come unexpectedly in terms of site-bond correlated-percolation theory. In general, this occurrence raises the question as to whether the spontaneous onset of regions of higher and lower polymer concentration (spinodal separation) may be regarded as a novel path for biomolecular interactions and the self-assembly of order in biomolecular systems.     

The influence of divalent cations on the dynamic properties of actin filaments: a spectroscopic study.

Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer.     

Using light scattering measurements to study the effects of monovalent and divalent cations on alginate aggregates

Light scattering measurements were used to assess the effectsof selected divalent and monovalent cations on alginate aggregationin vitro. Alginate, formed with either strontium, calcium orcobalt was partially dissolved with sodium. Calcium-alginatewas also partially dissolved with two other monovalent cations,lithium and potassium. Phosphate, when added to a solution containingcalcium-alginate, scrubbed algin-ate-bound calcium as well asfree calcium in solution. These findings provide an explanationfor an alternative approach for breaking down cell wall alginate. Key words: Alginate aggregates, monovalent cations, divalent cations, light scattering     

Differential stability of DNA crossovers in solution mediated by divalent cations

The assembly of DNA duplexes into higher-order structures plays a major role in many vital cellular functions such as recombination, chromatin packaging and gene regulation. However, little is currently known about the molecular structure and stability of direct DNA–DNA interactions that are required for such functions. In nature, DNA helices minimize electrostatic repulsion between double helices in several ways. Within crystals, B-DNA forms either right-handed crossovers by groove–backbone interaction or left-handed crossovers by groove–groove juxtaposition. We evaluated the stability of such crossovers at various ionic concentrations using large-scale atomistic molecular dynamics simulations. Our results show that right-handed DNA crossovers are thermodynamically stable in solution in the presence of divalent cations. Attractive forces at short-range stabilize such crossover structures with inter-axial separation of helices less than 20 Å. Right-handed crossovers, however, dissociate swiftly in the presence of monovalent ions only. Surprisingly, left-handed crossovers, assembled by sequence-independent juxtaposition of the helices, appear unstable even at the highest concentration of Mg²⁺studied here. Our study provides new molecular insights into chiral association of DNA duplexes and highlights the unique role divalent cations play in differential stabilization of crossover structures. These results may serve as a rational basis to understand the role DNA crossovers play in biological processes.     

Interaction of alginate single-chain polyguluronate segments with mono- and divalent metal cations: a comparative molecular dynamics study

The interaction of a set of monovalent (Na⁺, K⁺) and divalent (Mg²⁺, Ca²⁺) metal cations with single-chain polyguluronate (periodic chain based on a dodecameric repeat unit, 2₁-helical conformation) is investigated using explicit-solvent molecular dynamics simulations (at 300 K and 1 bar). A total of 14 (neutralising) combinations of the different ions are considered (single type of cation or simultaneous presence of two types of cation, either in the presence or absence of chloride anions). The main observations are: (1) the chain conformation and intramolecular hydrogen bonding is insensitive to the counter-ion environment; (2) the binding of the cations is essentially non-specific for all ions considered (counter-ion atmosphere confined within a cylinder of high ionic density, but no well-defined binding sites); (3) the density and tightness of the distributions of the different cations within the counter-ion atmosphere follow the approximate sequence Ca²⁺>Mg²⁺>K⁺～Na⁺; (4) the solvent-separated binding of the cations to the carboxylate groups of the chain is frequent, and its occurrence follows the approximate sequence K⁺>Na⁺>Ca²⁺>Mg²⁺ (contact-binding events as well as the binding of a cation to multiple carboxylate groups are very infrequent); and (5) the counter-ion atmosphere typically leads to a complete screening of the chain charge within 1.0–1.2 nm of the chain axis and, for most systems, to a charge reversal at about 1.5 nm (i.e. the effective chain charge becomes positive at this distance and as high in magnitude as one-quarter of the bare chain charge, before slowly decreasing to zero). These findings agree well (in a qualitative sense) with available experimental data and predictions from simple analytical models, and provide further insight concerning the nature of alginate–cation interactions in aqueous solution.     

Preparation of phenylalanine imprinted polymer by the sol–gel transition method

A phenylalanine (Phe) imprinted polymer was prepared by the wet-phase inversion and sol–gel transition method to endow a copolymer matrix with a large uptake capacity of template molecules and prominent adsorption selectivity at the high concentration of the racemate solution. A copolymer bead prepared by wet-phase inversion was shrunken in a hydrochloric acid solution containing a large amount of template molecules after swelling in a sodium hydroxide solution. Template molecules were effectively implanted in the polymer matrix during shrinking after swelling. The adsorption selectivities of Phe-imprinted copolymer bead were 2.1 and 1.33 at 1 g and 10 g Phe/l racemate solution, respectively, and the Phe uptake capacity reached about 1 g Phe/g dry weight of the copolymer. The adsorption selectivity of the copolymer was retained after five batches of adsorption/desorption in 1 g Phe/l solution composed of 5% D-Phe and 95% L-Phe.     

Structure of alginate gels: interaction of diuronate units with divalent cations from density functional calculations

The complexation of (1→4) linked α-L-guluronate (G) and β-D-mannuronate (M) disaccharides with Mg(2+), Ca(2+), Sr(2+), Mn(2+), Co(2+), Cu(2+), and Zn(2+) cations have been studied with quantum chemical density functional theory (DFT)-based method. A large number of possible cation-diuronate complexes, with one and two GG or MM disaccharide units and with or without water molecules in the inner coordination shells have been considered. The computed bond distances, cation interaction energies, and molecular orbital composition analysis revealed that the complexation of the transition metal (TM) ions to the disaccharides occurs via the formation of strong coordination-covalent bonds. On the contrary, the alkaline earth cations form ionic bonds with the uronates. The unidentate binding is found to be the most favored one in the TM hydrated and water-free complexes. By removing water molecules, the bidentate chelating binding also occurs, although it is found to be energetically less favored by 1 to 1.5 eV than the unidentate one. A good correlation is obtained between the alginate affinity trend toward TM cations and the interaction energies of the TM cations in all studied complexes, which suggests that the alginate affinities are strongly related to the chemical interaction strength of TM cations-uronate complexes. The trend of the interaction energies of the alkaline earth cations in the ionic complexes is opposite to the alginate affinity order. The binding strength is thus not a limiting factor in the alginate gelation in the presence of alkaline earth cations at variance with the TM cations.     

Selective transport of divalent cations by transition metal permeases: the Alcaligenes eutrophus HoxN and the Rhodococcus rhodochrous NhlF

nhlF and hoxN, the genes encoding a cobalt transporter of Rhodococcus rhodochrous J1 and a nickel permease of Alcaligenes eutrophus H16, respectively, were expressed in Escherichia coli. ⁵⁷Co²⁺ and ⁶³Ni²⁺ transport of the recombinants was examined by means of a previously described physiological assay. Although the transporters are highly similar, different preferences for divalent transition metal cations were observed. HoxN was unable to transport ⁵⁷Co²⁺, but mediated ⁶³Ni²⁺ uptake. The latter activity was unaffected by a tenfold excess of other divalent cations, showing the specificity of HoxN for Ni²⁺. In contrast, NhlF transported both ⁵⁷Co²⁺ and ⁶³Ni²⁺ ion. NhlF-mediated ⁶³Ni²⁺ uptake was markedly reduced in the presence of Co²⁺, while ⁵⁷Co²⁺ uptake was only slightly lower in the presence of Ni²⁺. These results indicate different affinities of NhlF for Co²⁺ and Ni²⁺ and identified Co²⁺ ion as the preferred substrate. Received: 8 September 1998 / Accepted: 30 November 1998     

Rheological and calorimetric study of the sol–gel transition of κ-carrageenan

Rheological and DSC techniques were used to study the effect of κ-carrageenan and KCl concentrations, 0–300 mM, on the sol–gel transition as well as on the linear viscoelasticity, at 25 °C, of the resulting gels. In heating and cooling DSC tests, the peak temperature was taken as the sol–gel transition point. In rheological tests, sol–gel transitions were determined from the variation of dynamic moduli with frequency and temperature, the independence of the phase angle on frequency and the evolution with temperature of dynamic moduli on cooling and heating at constant frequency and strain. Transition temperatures from DSC and rheology were in good agreement among them and with those previously reported. The three procedures yielded similar results, but the transition temperatures were more easily determined through the independence of the phase angle on frequency. Frequency sweeps showed gel behavior with stiffness increasing with polysaccharide and salt concentration. Below 100 mM KCl, G′ increased notably, whereas higher concentrations produced only marginal increases.     

The complexation of metal cations by D-galacturonic acid: a spectroscopic study

Solid complexes of D-galacturonic acid (GalA) with cobalt(II), copper(II), nickel(II) and oxovanadium(IV) (1-4) were prepared and characterised. The metal-to-ligand molar ratio was 1:2 for complexes 1-3 and 1:1 for complex 4. The alpha- and beta-anomers of GalA were detected in all the complexes in solid state and in solutions. An addition of small amounts of the paramagnetic complexes to the D2O solution of pure ligand led to NMR line broadening of some 1H and 13C nuclei. This broadening was sensitive to the anomeric state of GalA in the case of complexes 1 and 4. NMR and vibrational spectroscopic data indicate the formation of carboxylate complexes of all the cations, while noncarboxylic oxygens are also involved into the metal bonding in some cases. VCD spectra of complexes 1-4 in D2O and Me2SO-d6 solutions confirm that GalA carboxylic group may participate in the formation of optically active species around the metal cation. Possible ways of GalA coordination by metal cations of this study were proposed and discussed.     

Membrane fusion and the lamellar-to-inverted-hexagonal phase transition in cardiolipin vesicle systems induced by divalent cations.

The polymorphic phase behavior of bovine heart cardiolipin (CL) in the presence of different divalent cations and the kinetics of CL vesicle fusion induced by these cations have been investigated. (31)P-NMR measurements of equilibrium cation-CL complexes showed the lamellar-to-hexagonal (L(alpha)-H(II)) transition temperature (T(H)) to be 20-25 degrees C for the Sr(2+) and Ba(2+) complexes, whereas in the presence of Ca(2+) or Mg(2+) the T(H) was below 0 degrees C. In the presence of Sr(2+) or Ba(2+), CL large unilamellar vesicles (LUVs) (0.1 microm diameter) showed kinetics of destabilization, as assessed by determination of the release of an aqueous fluorescent dye, which strongly correlated with the L(alpha)-H(II) transition of the final complex: at temperatures above the T(H), fast and extensive leakage, mediated by vesicle-vesicle contact, was observed. On the other hand, mixing of vesicle contents was limited and of a highly transient nature. A different behavior was observed with Ca(2+) or Mg(2+): in the temperature range of 0-50 degrees C, where the H(II) configuration is the thermodynamically favored phase, relatively nonleaky fusion of the vesicles occurred. Furthermore, with increasing temperature the rate and extent of leakage decreased, with a concomitant increase in fusion. Fluorescence measurements, involving incorporation of N-NBD-phosphatidylethanolamine in the vesicle bilayer, demonstrated a relative delay in the L(alpha)-H(II) phase transition of the CL vesicle system in the presence of Ca(2+). Freeze-fracture electron microscopy of CL LUV interaction products revealed the exclusive formation of H(II) tubes in the case of Sr(2+), whereas with Ca(2+) large fused vesicles next to H(II) tubes were seen. The extent of binding of Ca(2+) to CL in the lamellar phase, saturating at a binding ratio of 0.35 Ca(2+) per CL, was close to that observed for Sr(2+) and Ba(2+). It is concluded that CL LUVs in the presence of Ca(2+) undergo a transition that favors nonleaky fusion of the vesicles over rapid collapse into H(II) structures, despite the fact that the equilibrium Ca(2+)-CL complex is in the H(II) phase. On the other hand, in the presence of Sr(2+) or Ba(2+) at temperatures above the T(H) of the respective cation-CL complexes, CL LUVs rapidly convert to H(II) structures with a concomitant loss of vesicular integrity. This suggests that the nature of the final cation-lipid complex does not primarily determine whether CL vesicles exposed to the cation will initially undergo a nonleaky fusion event or collapse into nonvesicular structures.     

Thermodynamic analysis of sol–gel transition of gelatin in terms of water activity in various solutions

Sol–gel transition of gelatin was analyzed as a multisite stoichiometric reaction of a gelatin molecule with water and solute molecules. The equilibrium sol–gel transition temperature, T_t, was estimated from the average of gelation and melting temperature measured by differential scanning calorimetry. From T_t and the melting enthalpy, ΔH_sol, the equilibrium sol‐to‐gel ratio was estimated by the van't Hoff equation. The reciprocal form of the Wyman–Tanford equation, which describes the sol‐to‐gel ratio as a function of water activity, was successfully applied to obtain a good linear relationship. From this analysis, the role of water activity on the sol–gel transition of gelatin was clearly explained and the contributions of hydration and solute binding to gelatin molecules were separately discussed in sol–gel transition. The general solution for the free energy for gel‐stabilization in various solutions was obtained as a simple function of solute concentration. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 685–691, 2015.     

Regulation of the fibronectin receptor affinity by divalent cations

The cell surface receptor for fibronectin is a heterodimeric membrane protein that recognizes an Arg-Gly-Asp sequence in fibronectin and that requires cations such as Mg2+ or Ca2+ for binding to fibronectin. The divalent cation requirements of this receptor were analyzed by measuring attachment of receptor liposomes to ligand-coated surfaces in the presence of different cations. The most striking effect observed was produced by Mn2+, which increased the binding of the receptor liposomes to fibronectin 2-3-fold over their binding in buffers containing Ca2+ and Mg2+. The binding activities of two related adhesion receptors, the vitronectin receptor and platelet GP IIb-IIIa, were supported but not enhanced by Mn2+. Two observations suggest that Mn2+ can compete with Ca2+ for the same cation-binding sites of the receptor. First, Mn2+ could still enhance fibronectin receptor binding activity even in the presence of 10-fold higher concentrations of Ca2+ or Mg2+. Second, Mn2+ inhibited the binding of radioactive Ca2+ to the alpha subunit of the receptor. The increased fibronectin receptor activity in the presence of Mn2+ appeared to be due to an increase in the affinity of the receptor for the Arg-Gly-Asp sequence because a 110-kDa cell attachment fragment and a synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited liposome binding more effectively in the presence of Mn2+ than in the presence of Ca2+/Mg2+. The affinity for the peptide was affected more than the affinity for the fragment, indicating that Mn2+ also induces a change in receptor specificity. Increased receptor binding in the presence of Mn2+ was also apparent in affinity chromatography of the fibronectin receptor on the 110-kDa fibronectin fragment; Mn2+ improved the yield of the receptor 4-fold. Mn2+ similarly increased the number of receptor-fibronectin complexes in preparations analyzed by electron microscopy. These results show that exogenous influences can modulate the affinity and specificity with which the fibronectin receptor binds to its ligands.     

Infrared spectroscopic study of the gel to liquid-crystal phase transition in live Acholeplasma laidlawii cells

The temperature dependences of the infrared spectra of deuterium-labeled plasma membranes of live Acholeplasma laidlawii B cells and of the isolated plasma membranes demonstrate that the profiles of the gel to liquid-crystal phase transitions are very different. At temperatures within the range of the phase transition, the live mycoplasma is able to keep the "fluidity" of its plasma membrane at a much higher value than that of the isolated plasma membrane at the same temperature. The difference is particularly pronounced at and around the temperature of growth. Live Acholeplasma laidlawii, grown at 37 degrees C on a fatty acid depleted medium supplemented with myristic acid (C14:0), pentadecanoic acid (C15:0), or palmitic acid (C16:0), are highly "fluid"; i.e., at the temperature of growth, the fractional population of the liquid-crystalline phase is 95-100% at 37 degrees C, whereas in the case of the isolated plasma membranes the fractional population of the liquid-crystalline phase at 37 degrees C is only 58% (C14:0), 36% (C15:0), or 38% (C16:0).