Rheological studies of synergistic kappa carrageenan-carob galactomannan gels

The mechanical properties, Young's modulus and yield stress of different carob galactomannan-kappa carrageenan blends have been examined using an Instron testing machine as a function of the following parameters:

1. (1) Constant total polysaccharide concentration.

2. (2) Galactomannan concentration for a given carrageenan concentration.

3. (3) Carrageenan concentration for a given galactomannan concentration.

4. (4) Galactomannan molecular weight.

5. (5) Carrageenan molecular weight.

The properties of the binary gels were compared to the kappa carrageenan gels. From the results, two types of gel structure could occur: a coupled network defined by specific junction zones (and involving unsubstituted mannose residues of the galactomannan chains) and also a gel structure with both polysaccharides associating independently.     

Reexamining the egg-box model in calcium-alginate gels with X-ray diffraction

The structure of the Ca--alginate junction zones was investigated with X-ray scattering on gels prepared with different methods. Fiber diffraction reveals the popular egg-box model may not be the only possible structure for the junction zones. The (001) reflection, which should be extinguished due to 2/1 helical conformation in the egg-box model, was observed. This was further confirmed by the measurements on Ca--alginate gel beads prepared at different pH where large pieces were formed through a relatively slow process, which leads to a higher crystallinity and a more perfect ordering. The results suggest a 3/1 helical conformation is more proper for Ca--alginate gels formed slowly. This does not exclude the possibility for the 2/1 helical conformation in fast gelatinized Ca--alginate in which the 2/1 helix is a metastable form. Comparing the X-ray scattering results of the fresh, dehydrated, and rehydrated gels, a reversible aggregation of junction zones is found during dehydration and rehydration. The different stabilities of initial bonds and bonds formed during drying are speculated to be the contribution of MG block or short G blocks in the junction zones.     

Stereocontrolled Synthesis of an Octasaccharide Part of I-Active Glycolipids

The effects of glycerin and ethylene glycol on the elastic modulus and DSC thermograms of agarose and kappa-carrageenan gels were examined to clarify the relation between structure and properties. The elastic modulus of these gels as a function of the concentration of polyols increased up to a certain concentration and then decreased with increasing concentration of polyols. These polyols shifted the melting temperature of the gel to higher temperatures in kappa-carrageenan gels but to lower temperatures in agarose gels. The temperature dependence of elastic modulus was changed in opposite directions in agarose and kappa-carrageenan gels by the addition of polyols, and this is discussed on the basis of model consisting of junction zones which are connected by Langevin chains. It was suggested that the mean distance between junction zones became shorter in the presence of a small amount of polyols.     

Ionic and acid gel formation of epimerised alginates; the effect of AlgE4

AlgE4 is a mannuronan C5 epimerase converting homopolymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 resulted in different amounts of alternating sequences. Both ionically cross-linked alginate gels as well as alginic acid gels were prepared from the epimerised alginates. Gelling kinetics and gel equilibrium properties were recorded and compared to results obtained with the original non-epimerised alginates. An observed reduced elasticity of the alginic acid gels following epimerisation by AlgE4 seems to be explained by the generally increased acid solubility of the alternating sequences. Ionically (Ca(2+)) cross-linked gels made from epimerised alginates expressed a higher degree of syneresis compared to the native samples. An increase in the modulus of elasticity was observed in calcium saturated (diffusion set) gels whereas calcium limited, internally set alginate gels showed no change in elasticity. An increase in the sol-gel transitional rate of gels made from epimerised alginates was also observed. These results suggest an increased possibility of creating new junction zones in the epimerised alginate gel due to the increased mobility in the alginate chain segments caused by the less extended alternating sequences.     

Gelation of sugarbeet and citrus pectins using enzymes extracted from orange peel

Sugarbeet pectin is shown to form gels in the presence of calcium using an enzyme preparation extracted from orange peel. The gels were transparent and exhibited no syneresis. The mechanism of gelation is chain association arising from both lowered pectin solubility and from formation of a limited network of calcium-linked junction zones. The gelation reaction involves limited pectin demethoxylation, the release of acetate presumably from C-2 or C-3 of galacturonyl residues, and a decrease in pH. The enzymes responsible are pectinesterase (EC 3.1.1.11) and pectin acetylesterase. We suggest that the latter is a novel activity associated with triacetin acetylesterase (EC 3.1.1.6). The gels are compared to citrus pectin gels made in the same way.     

Conformations and interactions of pectins: II. Models for junction zones in pectinic acid and calcium pectate gels

Pectinic acid and calcium pectate gels condensed into uniaxially oriented fibers have been studied by X-ray diffraction. Although the diffraction patterns correspond to systems of only limited order, they show that both systems conserve the 1.3 nm axial period and 0.43 nm pseudo-period observed in sodium pectate. Pectinic acid further resembles sodium pectate in packing isometrically in a hexagonal net of side 0.84 nm. On the other hand, calcium pectate fibers contain the 1.2 nm lateral spacing observed in pectic acid. Speculative models for pectinic acid and calcium pectate have been developed. The former structure could be stabilized by hydrophobic binding from columns of methyl groups as well as by specific intermolecular hydrogen bonds. In the latter, the main interactions between pairs of chains could be bridges formed by calcium ions, which incorporate into their co-ordination shells two polyanion oxygen atoms from one chain and three from another. These model-building studies provide plausible visualizations of two different kinds of junction zones that may exist in pectic gels.     

Transient electric birefringence of agarose gels. II. Reversing electric fields and comparison with other polymer gels

The transient electric birefringence of low electroendosmosis (LE) agarose gels oriented by pulsed unidirectional electric fields was described in detail in Part I [J. Stellwagen and N. C. Stellwagen (1994), Biopolymers, Vol. 34, p. 187]. Here, the birefringence of LE agarose gels in rapidly reversing electric fields, similar in amplitude and duration to those used for field inversion gel electrophoresis, is reported. Symmetric reversing electric fields cause the sign of the birefringence of LE agarose gels, and hence the direction of orientation of the agarose fibers, to Oscillate in phase with the applied electric field. Because of long-lasting memory effects, the alternating sign of the birefringence appears to be due to metastable changes in gel structure induced by the electric field. If the reversing field pulses are equal in amplitude but different in duration, the orientation behavior depends critically on the applied voltage. If E < 7 V/cm, the amplitude of the birefringence gradually decreases with increasing pulse number and becomes unmeasurably small. However, if E > 7 V/cm, the amplitude of the birefringence increase more than 10-fold after ～ 20 pulses have been applied to the gel, suggesting that a cooperative change in gel structure has occurred. Because there is no concomitant change birefringence must be due to an increase in the number of agarose fibers and /or fiber bundles orienting in the lectric field, which in turn indicates a cooperatice breakdown of the noncovalent “junction zones” that corss-link the fibers in to the fgel matrix. The sign of the birefringence of LE agarose gels is always positive after extensive junction zone breakdown, indicating that the agarose fibers and fiber bundles preferentially orient parallel to the lectric field when they are freed from the constraints of the gel matrix. Three other gel-forming polymers, high electroendosmosis (HEEO) agarose (a more highly changed agarose), β-carrageenan (a stereoisomer of agarose), and polyacrylamide (a chemically corss-linked polymer) were alos studied in unidirectional and rapidly reversing electric fields. The birefringence of HEEO agarose backbone chain. The β-carrageenan gels exhibit variable orientation behavior in reversing electric fields, suggesting that its internal gel structure is not as tightly interconnected as that of agaroise gels. Both HEEO agarose and β-carrageenan gels exhibit a large increase in the amplitude of the birefringence with increasing pulse number when asymmetric reversing pulses > 7 V/cm are applied to the gels, suggesting that junction zone breakdown in a common feature of polysaccharide gels. Chemically cross-linked polyacrylamide gels exhibit very small birefringence signals, indicating that very little orientation occurs in pulsed lectric fields. The sign of the birefringence is independent of the polarity of the lectric field, as expected from the Kerr law, and normal orientation behavior is observed in reversing electric fields. Hence, the anomalous change in sign of the birefringence observed for agarose gels in reversing electric fields must be due to the metastable junction zones in the agarose gel matrix, which allow gel fiber rearrangements to occur. © 1994 John Wiley & Sons, Inc.     

Using SAXS to reveal the degree of bundling in the polysaccharide junction zones of microrheologically distinct pectin gels

The results of microrheological studies carried out on ionotropic pectin gels, particularly the manifest power law behavior observed at high frequencies, indicate that by using different assembly conditions gels can be formed in which the elementary network strands have different stiffnesses. It has been hypothesized that these differences reflect different network architectures, the extreme cases of which might be described as (i) dimeric calcium-chelating junction-zones of limited extent, linked by considerably longer, flexible, single-chain sections, or (ii) semiflexible bundles consisting of extensively aggregated dimeric junction zones that latterly become entangled and cross-linked. To test this hypothesis directly, microrheologically distinct pectin gels have been generated using different assembly modalities, in particular by using different concentrations of polymer and cross-linking ions and by contrasting the controlled-release of ions or ion-binding groups, and the resulting systems have been studied by small-angle X-ray scattering. The results straightforwardly reveal that gels that are clearly more semiflexible from a microrheological point-of-view contain larger scattering entities than those with a more flexible character. Furthermore, a more detailed interpretation of the scattering data with the aid of molecular modeling suggests that for the gels formed here those with a semiflexible microrheological signature consist predominantly of network filaments consisting of four or more chains, whereas those with a more flexible signature are predominantly single-chain sections linked by dimeric associations with no more that a few percent of the chains bundled to any higher extent. The ability to generate differing network architectures from the same polymer that fulfill different functional requirements, either in vivo in the plant cell wall, where pectin plays a crucial structural and mechanical role, or in vitro in a myriad of applications, makes these biomimetic biopolymer networks of considerable interest.     

Small-angle X-ray scattering and rheological characterization of alginate gels. 3. Alginic acid gels

Alginic acid gels were studied by small-angle X-ray scattering and rheology to elucidate the influence of alginate chemical composition and molecular weight on the gel elasticity and molecular structure. The alginic acid gels were prepared by homogeneous pH reduction throughout the sample. Three alginates with different chemical composition and sequence, and two to three different molecular weights of each sample were examined. Three alginate samples with fractions of guluronic acid residues of 0.39 (LoG), 0.50 (InG), and 0.68 (HiG), covering the range of commercially available alginates, were employed. The excess scattering intensity I of the alginic acid gels was about 1 order of magnitude larger and exhibited a stronger curvature toward low q compared to ionically cross-linked alginate. The I(q) were decomposed into two components by assuming that the alginic acid gel is composed of aggregated multiple junctions and single chains. Time-resolved experiments showed a large increase in the average size of aggregates and their weight fraction within the first 2 h after onset of gelling, which also coincides with the most pronounced rheological changes. At equilibrium, little or no effect of molecular weight was observed, whereas at comparable molecular weights, an increased scattering intensity with increasing content of guluronic acid residues was recorded, probably because of a larger apparent molecular mass of domains. The results suggest a quasi-ordered junction zone is formed in the initial stage, followed by subsequent assembling of such zones, forming domains in the order of 50 A. The average length of the initial junction zones, being governed by the relative fraction of stabilizing G-blocks and destabilizing alternating (MG) blocks, determines the density of the final random aggregates. Hence, high-G alginates give alginic acid gels of a higher aggregate density compared to domains composed of loosely packed shorter junction zones in InG or LoG system.     

Steady-state and pulsed NMR studies of gelation in aqueous agarose

Steady-state and pulsed NMR techniques have been used to investigate molecular motion in sols and gels of agarose. In passing through the sol–gel transition, the molecular mobility of water molecules is reduced only by a small amount, whereas motion of the polymer chains is greatly attenuated. The results are discused in terms of the network theory of gelation, with references to the role of water in the process and the nature of the “junction zones” between polymer chains. T₂ and line-width measurements are dominated by exchange broadening. The effects of exchange rate and differences in relaxation time between the exchanging sites are discussed. The temperature hysteresis behavior of agarose gels has been investigated and the effects of “ageing” correlated with changes in nuclear relaxation times. The synergistic increase in gel strength obtained on adding locust bean gum (LBG) to agarose has been investigated. The results indicate that LBG does not form double-helix junctions and may decrease rates of gelation by steric effects. At high agarose concentration, the LBG remains mainly in solution in interstitial water, but at low agarose concentration, it is suggested that the LBG can link gel aggregates together into a self-supporting structure, producing a synergistic increase in gel strength. Comparisons have been made between the nature of the agarose–LBG interaction and agarose–cellulose interactions in biological systems.     

Effects of Potassium Chloride and Sodium Chloride on the Thermal Properties of Gellan Gum Gels

The exothermic and endothermic peaks in cooling and heating curves of differential scanning calorimetry (DSC) for gellan gum gels without and with potassium chloride and sodium chloride were analyzed. The gelling and melting temperatures shifted to higher temperatures with increasing gellan and salt concentration in the concentration range of gellan from 0.3 to 2.0% (w/w). The exothermic and endothermic enthalpy increased with increasing gellan and salt concentrations. Cooling DSC curves showed one exothermic peak for samples with salts and at low gellan concentration. Heating DSC curves showed many peaks for all samples except 0.3% (w/w) gellan gum gels. The sol-gel transition of samples was examined numerically by using a zipper model approach. The introduction of cations increases the number of junction zones or zippers and decreases the rotational freedom of parallel links. This makes the structure of junction zones more heat resistant, and increases the elastic modulus of the gel.     

Molecular transforms of kappa carrageenan and furcellaran from mixed gel systems

X-ray fibre diffraction studies of furcellaran-carob, furcellaran-tara, and furcellaran-konjac mannan mixed gels have failed to reveal any evidence for the predicted intermolecular binding between the algal polysaccharide helix and the galactomannan or glucomannan (konjac) mannan). In the absence of such interactions, mixed gels of kappa carrageenan-konjac mannan and furcellaran-konjac mannan, have been used to obtain good quality molecular transforms of the kappa carrageenan and furcellaran molecules in an oriented nematic liquid crystalline form. Analyses of the pattern support double helix structures with threefold symmetry with helix pitch of 2.5 nm. The absence of a 0.83 nm meridional in kappa carrageenan necessitates zero axial translation from the exact half-stagger position, contrary to the model building prediction. An axial translation from half-stagger is necessary for furcellaran.     

Chain-chain interactions for methyl polygalacturonate: models for high methyl-esterified pectin junction zones

The ability of pectins to form gels in the presence of calcium is well-known, and it implies the interaction of carboxylate groups and bivalent ions. However, even when most of the galacturonic units are methyl esterified, pectins are able to form gels but only under certain experimental conditions. In this case, hydrogen bonding and hydrophobic interactions are believed to be responsible for gel formation, and it is likely, as in the other mechanisms of polysaccharide gel formation, that stable junction zones consist of cooperatively ordered chains linked together throughout nonbonded interactions to provide a three-dimensional network. To investigate the junction zones in HM-pectin gels, we investigated, by molecular modeling, all of the ways to associate two, and then three, fully methyl-esterified galacturonic acid chains. Two models are obtained: the first one is based on a packing of parallel chains; it agrees with the hypothetical model derived from fiber diffraction study; the second one displays an antiparallel orientation of the chains; it presents a better arrangement of the chains and, theoretically, a much lower potential energy. In both cases, all of the favorable associations occur within a network of hydrogen bonds and of hydrophobic contacts.     

Rheological studies of specific cation forms of kappa carrageenan gels

Measurements have been made of the shear modulus of calcium, potassium and sodium kappa carrageenate gels as a function of polysaccharide concentration and temperature. Under the experimental conditions used the efficiency of the cations in gelling the polysaccharide has been found to be Ca²⁺>K⁺>Na⁺. The relative gelling efficiencies of the cations is attributed to their extent of hydration which controls the solubility of the salt form of the polysaccharide. Gelation is attributed to ‘microcrystallite’ formation at localised sites on adjacent polysaccharide chains. The sharp decrease of the shear modulus on heating is attributed to localised melting of these ordered regions.     

Analysis of the rat liver gap junction protein: clarification of anomalies in its molecular size

The major gap junction polypeptide in most tissues has an apparent molecular mass of 27 kDa with a 47 kDa dimer present in junction-enriched fractions. However, a 54 kDa protein recognized by gap junction-specific antibodies has been reported and a complementary DNA (cDNA) sequence for both human and rat liver gap junctions codes for a 32 kDa protein. In this paper we show that these are all forms of the same gap junction protein that can be observed on SDS-polyacrylamide gels simply by varying the concentration of acrylamide in the gels. A 64 kDa dimer is also obtainable. Antibodies to the gap junction protein or to a synthetic peptide constructed to match the rat liver gap junction amino-terminal sequence recognize all of these forms. Under some conditions a 54 kDa dimer is 'preferred', explaining the presence of this species in whole tissue homogenate Western blots. These results clarify several controversies and indicate that the protein forming the gap junction channel probably undergoes no major post-translational modification as the cDNA sequence codes for a protein of molecular mass 32 kDa and this protein species and its 64 kDa dimer are demonstrable on SDS-polyacrylamide gels under appropriate conditions.     

Influence of thermal history on the stability of gelatin gels

Gelatin gel properties have been studied using three techniques. Optical rotation measurements have shown that the lower the ageing temperature, the faster the helix content increases but the lower the helix stability. Rheological measurements show that a small increase in temperature leads to a melting of some junction zones followed by a build up of new ones. By ageing the gel at two successive temperatures one can show the existence of two populations of junction zones with different thermal stabilities. The same result is shown on melting thermograms obtained by differential scanning calorimetry. All these results are consistent with the hypothesis of the presence of junction zones of various lengths, the thermostability of which being proportional to their lengths.     

Agarose-dextran gels as synthetic analogs of glomerular basement membrane: water permeability

Novel agarose-dextran hydrogels were synthesized and their suitability as experimental models of glomerular basement membrane was examined by measuring their Darcy (hydraulic) permeabilities (kappa). Immobilization of large dextran molecules in agarose was achieved by electron beam irradiation. Composite gels were made with agarose volume fractions (phi(a)) of 0.04 or 0.08 and dextran volume fractions (phi(d)) ranging from 0 to 0.02 (fiber volume/gel volume), using either of two dextran molecular weights (500 or 2000). At either agarose concentration and for either size of dextran, kappa decreased markedly as the amount of dextran was increased. Statistically significant deviations from the value of kappa for pure agarose were obtained for remarkably small volume fractions of dextran: phi(d) > or = 0.0003 for phi(a) = 0.04 and phi(d) > or = 0.001 for phi(a) = 0.08. The Darcy permeabilities were much more sensitive to phi(d) than to phi(a), and were as much as 26 times smaller than those of pure agarose. Although phi(d) was an important variable, dextran molecular weight was not. The effects of dextran addition on kappa were described fairly well using simple structural idealizations. At high agarose concentrations, the dextran chains behaved as fine fibers interspersed among coarse agarose fibrils, whereas, at low concentrations, the dextran molecules began to resemble spherical obstacles embedded in agarose gels. The ability to achieve physiologically relevant Darcy permeabilities with these materials (as low as 1.6 nm2) makes them an attractive experimental model for glomerular basement membrane and possibly other extracellular matrices.     

Structural characteristics and rheological properties of partially hydrolyzed oat β-glucan: the effects of molecular weight and hydrolysis method

Partial hydrolysates of (1→3)(1→4)-β- -glucan from oats were produced by three hydrolysis methods: acid, cellulase or lichenase. The molecular weights ranged from 31 000 to 237 000 g/mol. Six percent solutions of small molecular weight β-glucans formed elastic gels after 4 days at 4 °C whereas larger molecular weight β-glucans remained viscous liquids after 7 days. The melting temperature of the gels increased as they aged and the peak heat flow temperature, measured by differential scanning calorimetry, was 62±2 °C. Partial hydrolysates produced with cellulase, which was shown to preferentially cleave regions of the molecule with longer contiguous β-(1→4)-linked -glucopyranosyl units, tended to produce more elastic gels with stronger junction zones than partial hydrolysates produced with lichenase which cleaves the β-(1→4) glycosidic 3-o-substituted glucose links. This suggests that β-(1→3)-linked cellotriose sections of the polymer are probably the segments which form the junction zones in the gel network rather than cellulose-like segments.     

Hindered convection of macromolecules in hydrogels

Hindered convection of macromolecules in gels was studied by measuring the sieving coefficient (theta) of narrow fractions of Ficoll (Stokes-Einstein radius, r(s) = 2.7-5.9 nm) in agarose and agarose-dextran membranes, along with the Darcy permeability (kappa). To provide a wide range of kappa, varying amounts of dextran (volume fractions < or = 0.011) were covalently attached to agarose gels with volume fractions of 0.040 or 0.080. As expected, theta decreased with increasing r(s) or with increasing concentrations of either agarose or dextran. For each molecular size, theta plotted as a function of kappa fell on a single curve for all gel compositions studied. The dependence of theta on kappa and r(s) was predicted well by a hydrodynamic theory based on flow normal to the axes of equally spaced, parallel fibers. Values of the convective hindrance factor (K(c), the ratio of solute to fluid velocity), calculated from Theta and previous equilibrium partitioning data, were unexpectedly large; although K(c) < or = 1.1 in the fiber theory, its apparent value ranged generally from 1.5 to 3. This seemingly anomalous result was explained on the basis of membrane heterogeneity. Convective hindrances in the synthetic gels were quite similar to those in glomerular basement membrane, when compared on the basis of similar solid volume fractions and values of kappa. Overall, the results suggest that convective hindrances can be predicted fairly well from a knowledge of kappa, even in synthetic or biological gels of complex composition.