Screening for synergistic interactions in dilute polysaccharide solutions

A simple viscometric approach has been used to screen for binding interactions between different polysaccharides in very dilute solution where exclusion effects should be negligible. The method involves preparing stock solutions to approximately the same, low, viscosity (η_sp≈1), dialysing to identical ionic conditions, mixing in various proportions, and looking for departures from the initial common viscosity.

Mixtures of xanthan or de-acetylated xanthan with locust bean gum (LBG) or konjac glucomannan (KM) show massive enhancement of viscosity, as anticipated from the formation of synergistic gels at higher concentrations. However, no viscosity changes on mixing with LBG or KM were observed for other conformationally ordered bacterial polysaccharides (welan and rhamsan) or for alginate and pectin with sufficient Ca²⁺ to induce almost complete conversion to the dimeric ‘egg box’ form, demonstrating that conformational rigidity is not, in itself, sufficient for other polysaccharides to form heterotypic junctions with mannan or glucomannan chains.

Interactions of carrageenans with LBG appear to depend on both conformation and the extent of aggregation. Mixtures of LBG with K⁺ kappa carrageenan in 100mM KCl (which is known to promote extensive aggregation of double helices) gave erratic values for rotational viscosity and showed typical gel-like mechanical spectra under low-amplitude oscillation. Disordered carrageenans (K⁺ kappa in water and lambda in 100mM KCl) showed no evidence of interaction with LBG. Negative results were also obtained for iota carrageenan under ionic conditions believed to promote ordering without significant aggregation (100mM KCl). However, under conditions where limited aggregation might be expected (iota carrageenan in 90 mM CaCl₂; Me₄N⁺ kappa carrageenan in 150 mM Me₄NI), significant reductions in viscosity were observed on mixing with LBG, which may indicate some intermolecular association but without the formation of an extended network structure.     

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.     

Effect of locust bean gum and konjac glucomannan oh the conformation and rheology of agarose and κ-carrageenan

Mixing with locust bean gum (LBG) induces obvious gel-like character in very dilute solutions of K⁺ κ-carrageenan (< 0.01% w/w in 100 mM KCl). At higher concentration (0.085%), addition of LBG (0.036%) gives a shoulder on the high-temperature side of the DSC (differential scanning calorimetry) exotherm associated with the carrageenan disorder-order transition, with an accompanying increase in gelation temperature and enhancement in gel strength (storage modulus, G′). On substitution of LBG by konjac glucomannan (KM) the shoulder in DSC cornverts to a discernable peak. Van't Hoff analysis of optical rotation data indicates that the high-temperature thermal processes could arise from association of LBG or KM chains to the carrageenan double helix as it forms, with the main transition at lower temperature corresponding to ordering of surplus carrageenan. With K -carrageenan in the nongelling tetra-methylaminonium salt form, addition of LBG causes no delectable change in DSC; rheological enhancement at high concentration (1% w/w) is limited to development of a very tenuous network, and in dilute solution a decrease in viscosity is observed. Agarose shows only a very slight increase in the disorder-order transition temperature on addition of KM, and it shows no detectable change with LBG. These observations are interpreted as showing that efficient binding of mannan or glucomannan chains requires some aggregation of the algal polysaccharide helices, but that extensive aggregation restricts synergistic interaction by competition with heterotypic association. © 1995 John Wiley & Sons, Inc.     

Competitive inhibition of interchain interactions in polysaccharide systems

The scope and validity of competitive inhibition to explore specific intermolecular association in polysaccharide systems is demonstrated, using alginate, carrageenan and xanthan-galactomannan gels as models. Alginate gelation can be abolished almost completely in the presence of an equimolar concentration of short poly-l-guluronate chain segments, and weakened by short mixed sequences, while poly-d-mannuronate has little effect; this is consistent with previous evidence of the relative importance of these three block-types in interchain association. Carrageenan gels are weakened by the presence of short homologous segments only under conditions of limited helix-helix aggregation, where it is understood that direct interchain association through double helices contributes appreciably to total crosslinking. Gel formation by association of the ordered conformation of xanthan with poorly substituted mannan backbone regions of galactomannans is significantly inhibited by the addition of a galactomannan having fewer unsubstituted chain sequences, and which can therefore bind to xanthan without significant network formation. In general, therefore, gel formation through ordered junction zones of fixed stoichiometry is inhibited by chains that are capable of participation in one stable junction, but not in two or more, so that they occupy binding sites without contributing to the development of the network.     

Photon transmission study on swelling of kappa-carrageenan gels prepared in various concentrations

κ-Carrageenan gels prepared with various carrageenan concentrations in pure water were completely dried and then swelled in pure water. Photon transmission measurements were performed using a UV-Vis (UVV) spectrometer during the swelling of κ-carrageenan gels. Transmitted photon intensity, I_tr, increased exponentially as swelling time is increased for all gel samples. The behaviour of I_tr was interpreted by Monte-Carlo Simulation. The increase in I_tr was quantified by employing Li-Tanaka equation, from which time constants τ₁ and collective diffusion coefficients, D_o were determined for the gels in various carrageenan concentrations. Gravimetric and volumetric measurements were also carried out during swelling of gels. It is observed that gel with high carrageenan content possess more double helices and more lattice dislocations and swell slower than gels with low carrageenan content which may contain less double helices and less lattice imperfections. Increase in I_tr was interpreted by the homogeneous distribution of double helices in the carrageenan gel system.     

Spatial configuration of ordered polynucleotide chains. V. Conformational energy estimates of helical structure

Semiempirical potential energy functional used previously to account successfully for the mean-square unperturbed dimensions and nmr coupling constants of randomly coiling polynucleotides are used, after modifications, to account for base stacking and interstrand hydrogen bonding, and to evaluate the conformational energies of single- and double-stranded polynucleotide helices. Attention is focused upon the variety of A-genus helices with local backbone conformations resembling the known double-helical structures of RNA. Distinct structural differences between single- and double-stranded helices are predicted from the energy calculations. A second point of interest is the apparent failure of two conformationally identical left-handed polynucleotide chains to form a left-handed duplex. The third major observation of the study is the wide morphological variety of theoreticaly allowed right-handed polynucleotide duplexes. In addition to the familiar double helix stabilized by horizontal base stacking and hydrogen bonding, an unusual vertical double helix is predicted to form between complementary bases fixed in the unusual but not energetically forbidden high anti glycosyl conformation. Experimental results bearing upon the theoretical predictions are discussed.     

Cation specificity of the order—disorder transition in iota carrageenan: effects of kappa carrageenan impurities

The temperature dependence of the optical rotation of carrageenan segments (iota, kappa and iota/kappa mixtures) in different salt solutions (tetramethylammonium or potassium chloride) has been studied. In solutions of pure iota carrageenan (90% iota structure) no evidence of any pronounced cation specificity nor of any thermal hysteresis in the iota carrageenan order-disorder transition was found. However, mixtures of iota and kappa carrageenan displayed a cation specificity similar to that previously observed in iota samples of lesser chemical purity, indicating that kappa impurities in iota samples may be located in regular sequences, rather than randomly in the carrageenan primary structure. Our findings strongly suggest that the order-disorder transitions of iota and kappa sequences in mixed samples occur independently.     

The relationship between internal chain length of amylopectin and crystallinity in starch

Molecular models of amylopectin were created and investigated by computer simulation. First, single and double helices of various lengths were constructed. The 1 → 6 branching in double and single helices of amylopectin was studied. Subunits of single helices, double helices, and branch points were used as building blocks of larger systems. The possible makeup of amylopectin unit clusters was investigated via a series of models, including single–single, double–single, and double–double helix systems. The lengths of the single helix section that linked two branch points (internal chains) was systematically varied between values of 0–10 glucose residues. It was found that certain internal chain lengths lead to parallel double helices. Thus, it was postulated that the length of internal chains may determine the degree of local crystallinity. Furthermore, it was noted that some of the low‐energy arrangement of double helices could be superimposed on either the two adjacent and nonadjacent double helices of crystalline A and B starch polymorphs. In other cases, the distance between the double helices is so large that it may in fact be a model for branching between two amylopectin crystals or unit clusters. Results obtained through this work were corroborated, where possible, with information available from crystallographic, branching, and enzymatic studies. © 1999 John Wiley & Sons, Inc. Biopoly 50: 381–390, 1999     

DNA associations: packing calculations in A-, B-, and Z-DNA structures.

A detailed theoretical study has been carried out to examine the modes of DNA-DNA interactions on the basis of hard-sphere contact criteria. Two helices of identical structure and length are oriented side-by-side and their relative positions are controlled by translations along and rotations about specific axes. Short atomic contacts between pairs of atoms in the structures are assessed and contact-free configurations are compiled. The computed contact-free arrangements of A, B, and Z double helices are found to be remarkably similar to the packing motifs observed in DNA crystals and stretched fibers. Equally interesting in the study are the broad ranges of sterically acceptable arrangements that preserve the overall packing morphology of neighboring duplexes: Among the most notable morphological features in the helical complexes are extended "super" major and minor grooves which might facilitate the wrapping and packaging of DNA chains in supramolecular assemblies. The hard-sphere computations, however, are insufficient for quantitative interpretation of the packing of DNA helices in the solid state. The results are, nevertheless, a useful starting point for energy based studies as well as relevant to the analysis of long-range interactions in DNA supercoils and cruciforms.     

Molecular Modelling of Helix Stability in Carrageenans

Molecular models of disaccharides, and single and double helices up to eight monomers in length have been constructed of the two types of glycosidic linkage in the carrageenan chain. These links are a galactose to anhydrogalactose link (GA link), and an anhydrogalactose to galactose link (AG link). These models are also based on 3-carrageenan, which contains a 4-sulphate galactose ring. The effects of the sulphate groups on the conformation of the helices may be seen by the angles of $ϕ$ϕ and N explored during the simulations by the AG and GA linkages. It has been observed that the molecule can explore a greater area of conformational space about the GA link than the AG link. This could be due to steric hindrance caused by the bulky sulphate group near the AG link. The sulphate group is further away from the GA link than from the AG link, and this may provide a possible explanation for the relatively unhindered movement about the GA link compared to the AG link. The results have also shown that the conformational space for the AG linkages, as well as the GA linkages vary between different lengths of the polysaccharide chain. Single helix models show little stability in molecular dynamics simulation, whereas the eight monomer double helix model is more stable than a six monomer double helix model.     

The spatial configuration of ordered polynucleotide chains. II. The poly(rA) helix.

Approximate details of the spatial configuration of the ordered single-stranded poly(rA) molecule in dilute solution have been obtained in a combined theoretical analysis of base stacking and chain flexibility. Only those regularly repeating structures which fulfill the criterion of conformational flexibility (based upon all available experimental and theoretical evidence of preferred bond rotations) and which also exhibit the right-handed base stacking pattern observed in nmr investigations of poly(rA) are deemed suitable single-stranded helices. In addition, the helical geometry of the stacked structures is required to be consistent with the experimentally observed dimensions of both completely ordered and partially ordered poly(rA) chains. Only a single category of poly(rA) helices (very similar in all conformational details to the individual chains of the poly(rA) double-stranded X-ray structure) is thus obtained. Other conformationally feasible polynucleotide helices characterized simply by a parallel and overlapping base stacking arrangement are also discussed.     

The structure of aquaporin-1 at 4.5-A resolution reveals short alpha-helices in the center of the monomer

Aquaporin-1 is a water channel found in mammalian red blood cells that is responsible for high water permeability of its membrane. Our electron crystallographic analysis of the three-dimensional structure of aquaporin-1 at 4.5-A resolution confirms the previous finding that each subunit consists of a right-handed bundle of six highly tilted transmembrane helices that surround a central X-shaped structure. In our new potential map, the rod-like densities for the transmembrane helices show helically arranged protrusions, indicating the positions of side chains. Thus, in addition to the six transmembrane helices, observation of helically arranged side-chain densities allowed the identification of two short alpha-helices representing the two branches of the central X-shaped structure that extend to the extracellular and cytoplasmic membrane surfaces. The other two branches are believed to be loops connecting the short alpha-helix to a neighboring transmembrane helix. A pore found close to the center of the aquaporin-1 monomer is suggested to be the course of water flow with implications for the water selectivity.     

Alpha-hairpin stability and folding of transmembrane segments

Molecular Dynamics (MD) simulations at low dielectric constant have been carried out for peptides matching the double spanning segments of transmembrane proteins. Different folding dynamics have been observed. The peptides folded into the stable helix-turn-helix conformation-alpha-hairpin-with antiparallel-oriented strands or unstable alpha-hairpin conformation that unfolded later into the straight helical structure. The peptide having flexible residues in the TM helices often misfolded into a tangled structure that can be avoided by restricting the flexibility of these residues. General conclusions can be drawn from the observed folding dynamics. The stability and folding of some double spanning transmembrane fragments are self-assembling. The following and/or neighboring peptide chains of the protein may support the stability of the hairpin structure of other fragments. The stability of the TM helices containing flexible residues could be maintained due to contacts with neighboring TM segments.     

Mode of assembly of amphipathic helical segments in model high-density lipoproteins

The structure of discoidal apo A-I-phospholipid complexes, representing the metabolic precursors of mature high-density lipoprotein particles, was studied by a combination of both a theoretical and an experimental approach. The secondary structure of the complex was determined by circular dichroic measurements, while the relative orientation of the apo A-I helical segments and of the phospholipid acyl chains was determined by ATR infrared measurements. Fluorescence energy transfer between the tryptophan residues of apo A-I and fluorescent phospholipid probes yielded an estimation of the relative topography of the lipid and apolipoprotein components in discoidal and spherical particles. The theoretical approach consisted of the identification of the helical segments in various apo A-I species. These segments were then oriented at a lipid/water interface by minimization of their hydrophobic and hydrophilic transfer energies. The calculation of the hydrophobicity profiles along the axis of the helices leads to the identification of specific interactions between pairs of helices. The helices were further assembled together with the phospholipids by computer modelling, enabling an estimation of the dimensions of the complex. The combination of the experimental and theoretical results yielded a model for discoidal apolipoprotein-phospholipid complexes, in which the amphipathic helical segments are oriented along the edges of the discs. Such a model can be extended to the conversion of these complexes into mature spherical HDL, through the formation of a cholesteryl ester core.     

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.     

Origin of the limited alpha-amylolysis of debranched maltodextrins crystallized in the A form: a TEM study on model substrates

The detailed ultrastructure of a new type of resistant starch and the way that it is modified during hydrolysis by alpha-amylases were studied by transmission electron microscopy (TEM) on model starch crystals. The selected substrates were waxy maize starch lintners and A-type crystals prepared from low degree of polymerization (DP) amylose. A model describing the stacking of double helices is proposed for A-type low DP amylose crystals. The enzymatic hydrolysis of both lintners and low DP crystals has been shown to occur by the side of double helices and not their ends. The results were transposed to a new type of resistant starch (RS) produced by debranching maltodextrins in concentrated solutions. This product presents A-type crystallinity contrary to all other known classified RS. Moreover it consists of low DP chains similar to the model crystals studied and yields similar electron diffraction patterns to those of A-type low DP crystals. The similarities in the morphology of these substrates with that of the studied RS led us to attribute its resistance to its particularly dense and compact morphology, resulting from the epitaxial growth of elementary crystalline A-type platelets. In the resulting structure, the accessibility of double helices to alpha-amylase is strongly reduced by aggregation.     

Assignment of antigenic determinants to separated I-A kappa chains

The alpha- and beta-chains of the I-A kappa antigen from the AKTB-1b B cell lymphoma were separated by ion-exchange chromatography on CM-Sephadex in the presence of propionic acid and urea. Removal of the denaturants by dialysis produced isolated chains that regained a significant amount of their native configuration. These materials were used with a battery of monoclonal antibodies in a direct binding assay to localize specific alloantigenic determinants to the A alpha kappa or A beta kappa chains. This method allowed the assignment of the nominal specificity Ia. 17 and at least one epitope of the specificity Ia.2 to the A beta kappa chain. Finally, the I-A kappa antigen from the B cell lymphoma AKTB-1b was shown to be identical, by the criterion of tryptic peptide analysis, to that derived from normal B10.BR splenocytes. This constitutes the first demonstration that the polypeptide portion of a tumor-derived class II MHC antigen is identical to that derived from a normal tissue.     

The influence of locust bean gum and dextran on the gelation of κ-carrageenan

The interaction of κ-carrageenan with locust bean gum and dextran has been studied by rheology, differential scanning calorimetry (DSC), and electron spin resonance spectroscopy (ESR). Rheological measurements show that the carrageenan gel characteristics are greatly enhanced in the presence of locust bean gum but not in the presence of dextran. Carrageenan/locust bean gum mixtures show two peaks in the dsc cooling curves. The higher temperature peak corresponds to the temperature of gelation and its intensity increases at the expense of the lower temperature peak as the proportion of locust bean gum in the mixture increases. Furthermore, the DSC heating curves show enhanced broadening when locust bean gum is present, indicating increased aggregation. These results are taken as evidence of carrageenan/locust bean gum association. The gelation process has also been followed by ESR using spin-labeled carrageenan. On cooling carrageenan solutions, an immobile component appears in the ESR spectra signifying a loss of segmental mobility consistent with chain stiffening due to the coil → helix conformational transition and helix aggregation. For carrageenan/locust bean gum mixtures, carrageenan ordering occurs at temperatures corresponding to the higher temperature DSC setting peak and the temperature of gelation. Similar studies using spin-labeled locust bean gum show that its mobility remains virtually unaffected during the gelation process. It is evident, therefore, that carrageenan and locust bean gum interact only weakly. It is proposed that at low carrageenan concentrations the gel network consists of carrageenan helices cross-linked by locust bean gum chains. At high carrageenan concentrations the network is enhanced by the additional self-aggregation of the “excess” carrageenan molecules. For carrageenan/dextran mixtures, only one peak is observed in the dsc cooling curves. The onset of gelation shifts to higher temperatures only at very high (20%) dextran concentrations and this is attributed to volume exclusion effects. Furthermore, there is no enhanced broadening of the peaks in the DSC heating curves as for the carrageenan/locust bean gum systems. It is therefore concluded that carrageenan/dextran association does not occur. The difference in behavior between locust bean gum and dextran is attributed to the greater flexibility of the dextran chains. © 1996 John Wiley & Sons, Inc.     

MONOCLONAL ANTIBODIES AS MOLECULAR MARKERS FOR THE INTRACELLULAR AND CELL WALL DISTRIBUTION OF CARRAGEENAN EPITOPES IN KAPPAPHYCUS (RHODOPHYTA) DURING TISSUE DEVELOPMENT1

Carrageenan, the major cell wall carbohydrate of certain red algae, is variable in structure and gelling properties. Sequence types include gelling (kappa and iota) and nongelling (lambda) types in addition to precursors, often in hybrid molecules containing more than one precursor and/or sequence type. Molecular markers to subunits were needed to study carrageenan synthesis, cell wall organization, and the relationship between structure and function. Monoclonal antibodies were produced to carrageenan, and their specificities were determined by competitive enzyme immunoassay. Antibodies were identified with specificities related to kappa, iota, and lambda carrageenan. The patterns of immunofluorescence localization on Kappaphycus alvarezii = Eucheuma alvarezii var. tambalang (Doty) sections were distinctive for each antibody. The antibody to a kappa-related epitope labeled mature tissue strongly; antibodies to an iota-related epitope and a lambda-related epitope labeled weakly, consistent with the kappa-enriched carrageenan produced by this alga. Kappa-related epitopes were distributed throughout the wall and matrix, whereas iota-related epitopes were concentrated in the middle lamella. Lambda-related epitopes were localized primarily at the plant cuticle where kappa and iota antigens were lacking. An antibody appeared to be specific for a precursor of the gelling subunits because it showed maximal wall and intracellular labeling at the youngest developmental stage. All antibodies labeled intracellular inclusions in the transition zone between the epidermis and medulla during the development of medullary cells from the peripheral meristem in young branches. The results demonstrate the intracellular synthesis of epitopes related to all major carrageenan subunits and their differential extracellular distribution.