Electron microscopy of native xanthan and xanthan exposed to low ionic strength

Optical rotation data indicate that xanthan can exist both in an ordered and a disordered conformation. Using molecular weights obtained from light scattering measurements and contour length distributions obtained from electron micrographs, we find that a native, filtered xanthan exposed to low salinity (< 10^?4M NaCl) and subsequently returned to 0.1M NaCl has a highly elongated structure with a mass per unit length of 1950 ± 200 Dalton/nm. Our data thus suggest that the ordered conformation of this xanthan is double stranded. We find that native, filtered xanthan in 0.1M NH₄Ac has a nearly similar structure, but exists in part as aggregates of varying shape and size. Electron micrographs of these xanthans in 10^?4M NH₄Ac (the disordered conformation) display a mixture of species ranging from unaggregated single- or perfectly matched double-stranded species, to double-stranded chains branching into its two subunits as well as double-stranded chains with different degrees of mismatching. This study suggests that the perfectly matched antiparallel or parallel double-stranded chain constitutes the lowest free energy state of the ordered conformation of xanthan in dilute aqueous solution.     

Revisiting the conformation of xanthan and the effect of industrially relevant treatments

The structure and conformation of xanthan in aqueous solution following various processing treatments typically encountered in its application were investigated in this study. Treatments such as heating, autoclaving, high pressure homogenisation and irradiation were subjected to the same sample. Parameters such as weight average molecular weight (M(w)), polydispersity index, root mean square radius of gyration, intrinsic viscosity and Huggins constant were used to monitor the effect of these treatments. Additionally, we have quantified the mass recovery of samples examined by gel permeation chromatography and light scattering to properly account for all fractions present in xanthan solutions. Atomic force microscopy (AFM) images together with height measurements confirmed that xanthan conformation is double helical ordered renatured state (pre-heat treated by the manufacturer) in dilute solution conditions and random coil conformation in very dilute solution. The ordered (renatured) conformation is shown to have partially molten double helix, with more flexibility than the perfectly ordered native double helix. Heat treatment for 2h at 85°C reduces the M(w) of xanthan to half its initial value, and mass recovery measurements indicate that it completely overcomes its associative nature. Thermally treated xanthan solution in the dilute region leads to an order-disorder transition, as determined by contour length per unit mass. Similarly, irradiation of xanthan solution results in an order-disorder transition together with the production of single strand low molecular weight molecules. Autoclaving and high pressure homogenisation treatments cause degradation of xanthan. The results from treated xanthan solutions following high pressure homogenisation and irradiation confirm that xanthan does not reassociate. A revised summary of xanthan conformation in solution together with schematic models following the various treatments are proposed.     

Conformation of xanthan dissolved in aqueous urea and sodium chloride solutions

Quasielastic light-scattering and other physical-chemical techniques have been used to compare the conformation and intermolecular interactions of xanthan in water, aqueous sodium chloride, and urea solutions. The results showed that xanthan dissolved in 4m urea has a disordered conformation after the solution has been maintained for 3 h at 95° and then cooled to room temperature. This conformation is similar to that previously observed only in solutions having low ionic strength at higher temperatures, following disruption of the ordered, low-temperature form. “Anomalous” behavior is seen for xanthan as a function of ionic strength, in that the hydrodynamic radius increases with increase in ionic strength, whereas a decrease is typical for polyelectrolytes. These observations suggest that aggregation of rod-like chains, similar to that seen for other stiff-chain polymers, occurs for xanthan in salt solutions, where the charged groups of the polyelectrolyte are screened by the salt ions. This aggregation may explain some of the high values reported in the literature for the molecular weight.     

“Melt-in-the-mouth” gels from mixtures of xanthan and konjac glucomannan under acidic conditions: A rheological and calorimetric study of the mechanism of synergistic gelation

The effect of acidification on a typical commercial xanthan and on pyruvate-free xanthan (PFX), alone and in gelling mixtures with konjac glucomannan (KGM), has been studied by differential scanning calorimetry (DSC) and small-deformation oscillatory measurements of storage modulus (G′) and loss modulus (G″). For both xanthan samples, progressive reduction in pH caused a progressive increase in temperature of the disorder–order transition in DSC, and a progressive reduction in gelation temperature with KGM. This inverse correlation is interpreted as showing that synergistic gelation involves disruption of the xanthan 5-fold helix, probably by attachment of KGM to the cellulosic backbone of the xanthan molecule (as proposed previously by a research group in the Institute of Food Research, Norwich, UK). Higher transition temperature accompanied by lower gelation temperature for PFX in comparison with commercial xanthan at neutral pH is explained in the same way. However, an additional postulate from the Norwich group, that attachment of KGM (or galactomannans) can occur only when the xanthan molecule is disordered, is inconsistent with the observation that gelation of acidified mixtures of KGM with PFX can occur at temperatures more than 60 °C below completion of conformational ordering of the PFX component (as characterised by DSC). Increase in G′ on cooling for mixtures of commercial xanthan with KGM at pH values of 4.5 and 4.25 occurred in two discrete steps, the first following the temperature-course observed for the same mixtures at neutral pH and the second occurring over the lower temperatures observed for mixtures of KGM with PFX at the same values of pH. These two “waves” of gel formation are attributed to interaction of KGM with, respectively, xanthan sequences that had retained a high content of pyruvate substituents, and sequences depleted in pyruvate by acid hydrolysis. At pH values of 4.0 and lower, gelation of mixtures of KGM with commercial xanthan followed essentially the same temperature-course as for mixtures with PFX, indicating extensive loss of pyruvate under these more strongly acidic conditions. Mixtures prepared at pH values in the range 4.0–3.5 gave comparable moduli at room temperature (20 °C) to those obtained at neutral pH, but showed substantial softening on heating to body temperature, suggesting possible applications in replacement of gelatin in products where “melt-in-the-mouth” characteristics are important for acceptability to the consumer.     

Salt-induced extension and dissociation of a native double-stranded xanthan

This study shows that xanthan molecules at room temperature may assume at least three different conformations in 0.1 m NaCl aqueous solutions in which the local structure is ordered: (1) the native compact double helix, (2) the extended double helix, and (3) the extended single helix. Experiments including viscosity, low-angle light scattering and optical rotation measurements have been carried out with a fully pyruvated and fully acetylated native laboratory sample supplied as fermentation broth. Two major conformation changes of the native double helix which were found irreversible in our experimental conditions can be induced by treatments at low ionic strength. After treatment in 10⁻⁴m NaCl, xanthan is still a double helix in 10⁻¹m NaCl, but the backbone of each strand has been extended. After the sample has been in 10⁻⁵m NaCl, the double helix has been dissociated and a single helix sample is obtained. Thus, the denaturing of xanthan is a two-step process. The first step consists of the extension of the two chains inside the double helix, and the second is a dissociation of the native double strand.     

Sodium and calcium counterion activity in the presence of xanthan polysaccharide

This work concerns the activity coefficient determination for sodium and calcium ions in a semi-dilute solution of xanthan when they are mixed in various ratios, and their correlation with the conformational structures. From these values we show that the conformation can be related to the dissociation of the polymer carboxylic acid sites. Below an apparent dissociation coefficient of about 0.29, the conformation is ordered and above 0.55 the xanthan conformation is entirely disordered. The value of the counterion activity coefficient in pure solution of sodium or calcium xanthan salts agrees fairly well with the theoretical ones derived from Manning's and Lifson-Katchalsky's theories if we make the assumption that the Na form is a stretched coil and the Ca form a single fivefold helix.     

Studies on the viscosity behavior of concentrated alkali halides in aqueous maltose solutions

The viscosities of concentrated solutions of sodium and potassium halides (concentration range 0.125 to 3.0m) have been measured in aqueous maltose solution at 25, 30, 35, and 40°. Various equations employed for concentrated solutions of electrolytes have been tested, to ascertain the validity of the relative viscosity data. In order to elucidate the structural behavior of sodium and potassium halides in aqueous maltose solution, the molar volumes ($\text{V}$), ionic B-coefficients, and hydration numbers (n_B) of various ions have been computed. The B₊ and B_? coefficients have been interpreted in terms of solute-solvent interactions. On the basis of the data, it has been found that, in 0.5m maltose solution, the different ions show structure-breaking tendency in the order: I^? > Br^? > Cl^? > K⁺ > Na⁺.     

Impact of the extrusion process on xanthan gum behaviour

Processing xanthan gum by extrusion and subsequent drying produces a biopolymer showing particulate, rather than molecular behaviour in aqueous solution. This form of xanthan disperses very readily to give a viscosity that is strongly dependent on salt concentration. On heating above the temperature of the order-disorder transition as determined by calorimetry, there is a viscosity transition that is indicative of the irreversible loss of the particulate structure. It is suggested that the extrusion process melts and aligns xanthan macromolecules. On cooling reordering will occur but in the highly concentrated environment in the extruder ( approximately 45% water w/w), inter-molecular association between neighbouring macromolecules cannot proceed to completion due to kinetic trapping. As a consequence a network structure is created maintained by associations involving ordered regions. A xanthan solution can be prepared from this particulate material by dispersing and subsequent heating far more readily than can be achieved with non-processed xanthan.     

Structure and chain conformation of five water-soluble derivatives of a β-d-glucan isolated from Ganoderma lucidum

A linear water-insoluble (1→3)-β-d-glucan, coded as GL-IV-I, was isolated from the fruit body of Ganoderma lucidum by extracting with NaOH solution. Its derivatives were prepared by using sulfation, carboxymethylation, hydroxyethylation, hydroxypropylation, and methylation, respectively, and these were labeled as S-GL, CM-GL, HE-GL, HP-GL and M-GL. Five derivatives exhibited good water solubility. Their structures and chain conformations were investigated with infrared spectroscopy, elemental analysis (EA), one- and two-dimensional NMR spectroscopy, laser light scattering (LLS), and size-exclusion chromatography combined with LLS (SEC-LLS). The reactivity of the hydroxyl group of GL-IV-I was ordered as C-6 > C-4 > C-2 for the five derivatives. The degree of substitution (DS) of the derivatives was calculated from EA and NMR spectroscopy to be from 0.32 to 1.18. The weight-average molecular mass (M_w) of GL-IV-I, S-GL, CM-GL, HE-GL, HP-GL, and M-GL was 13.3 × 10⁴, 10.1 × 10⁴, 6.3 × 10⁴, 7.2 × 10⁴, 5.1 × 10⁴, and 14.1 × 10⁴, respectively. The conformation analysis studies revealed that GL-IV-I exists as a compact coil in dimethyl sulfoxide, whereas the five derivatives are slightly expanded flexible chains in 0.9% aqueous NaCl solution.     

Enzymic hydrolysis of the bacterial polysaccharide xanthan by cellulase

In this paper the mechanism of enzymic hydrolysis by a cellulase on xanthan is investigated. It is demonstrated that in salt free solution there is a random breakdown of the main chain when the polysaccharide is in the unordered conformation. The apparent rate of hydrolysis followed by the decrease of the solution viscosity depends on the quality of the solution. In addition, the rate of hydrolysis may be directly correlated with the degree of local order expressed by its specific rotary power. It is shown that there is no hydrolysis on the ordered helical conformation.     

Depolymerization of xanthan by iron-catalysed free radical reactions

It is demonstrated using gamma-radiolytic and photolytic techniques that, when O₂?- is produced in the presence of catalytic amounts of iron(II) and iron(III) complexes depolymerization of xanthan in aqueous solution occurs. The use of these techniques also allows a quantitative measurement of the efficiency of conversion of O₂^?- to ?OH in these systems to be made, with the assumption that ?OH is the most likely depolymerizing species present. Using pulse radiolysis and oxidative-reductive depolymerization, the effects of some common anions on the degradation process have been correlated with the reactivities of their related free radicals with xanthan. The effects of other parameters such as ionic strength and temperature on xanthan depolymerization are also detailed     

Associations of like and unlike polysaccharides: Mechanism and specificity in galactomannans,interacting bacterial polysaccharides,and related systems

Chiroptical, rheological, and n.m.r.-relaxation evidence is presented, to identify interactions of two types between different polysaccharides: (1) mutual exclusion of incompatible molecules, with consequent increase in the effective concentration of both; and (2) energetically favourable association of structurally and sterically regular chain-segments. β-1,4-linked plant polysaccharides interact by association of unsubstituted backbone regions, either with like chians, or with sterically compatible, unlike molecules. Extracellular polysaccharides (xanthans) of Xanthomonas plant pathogens maintain their ordered native conformation in solution, and this accounts for their industrially valuable, rheological peculiarities. These materials bind strongly to the plant glycans. Random-coil bacterial gums show no such interactions, although dextran enhances autogelation of galactomannans by exclusion. Extracellular polysaccharides from Arthrobacter species also have ordered native conformations in solution, but do not share the specific interactions of xanthan. Native xanthan shows marked specificity in its interactions with plant glycans, indicating a possible biological role in host-pathogen recognition.     

The influence of thermal treatment and operational conditions on xanthan produced by X. arboricola pv pruni strain 106

The influence of thermal treatment and operational conditions (pH and stirrer speed) used in the process of xanthan production by Xanthomonas arboricola pv pruni strain 106 were evaluated through yield of xanthan, aqueous solution and fermentation broth viscosity, sodium content, pyruvate and acetyl content and molar mass. Different conditions used during the fermentation affected the xanthan characteristics. Thermal treatment decreased the final yield and pyruvate and acetyl content, and increased the xanthan aqueous solution and fermentation broth viscosities, as well as molar mass. In this study the best combination of yield and viscosity was obtained with the use of pH 7 and 400 rpm during fermentation and post-fermentation thermal treatment. Aggregation of xanthan molecules promoted by heating and detected through an increase of molar mass was apparently affected by the sodium content. As a result, a correlation between molar mass and xanthan solution viscosity could be observed.     

Activity coefficients of counterions and conformation in kappa-carrageenan systems

The optical rotation and the conductivity of kappa-carrageenans in aqueous solution have been investigated as functions of temperature in the presence of various electrolytes. The activity coefficients of sodium and potassium have been determined and correlated with the conformation. The potassium activity coefficient under ordered conformation is in agreement with a mechanism of dimerization.     

Raman spectral studies of nucleic acids. XI. Conformations of yeast tRNAPhe and E. coli ribosomal RNA in aqueous solution and in the solid state

Laser-excited Raman spectra of tRNA^Phe from yeast and of fractionated 16S and 23S rRNA from E. coli are reported for samples in aqueous solution and in the solid state. The Raman scattering spectrum of each RNA is not significantly altered by the change from an aqueous to a solid environment and displays the same characteristic frequencies and intensities associated with ordered polyribonucleotide structures. Unlike DNA, the backbone conformation of RNA thus appears to be largely insensitive to gross changes in the degree of hydration. Raman scattering from the phosphate group vibrations of aqueous tRNA_yeast^Phe is qualitatively and quantitatively the same as obtained from previously studied tRNA's and is indicative of a highly ordered conformational structure in which some 85% of the nucleotide residues are in ordered configurations. The major differences observed between spectra of tRNA and rRNA are attributed to differences in base composition of these RNA's.     

An Investigation of the Stereochemistry of some Nickel(II) Tetraamines in Acetonitrile Solution

The equilibria of adduct formation between several nickel(II) tetraamine complexes and acetonitrile were determined from −40 to 80 °C in acetonitrile solution by the Evans NMR magnetic susceptibility method. The stability order for adduct formation of the paramagnetic complex in terms of the ligand was found to be: 2,2,2-tet, 3,3,3-tet > 3,2,3- tet > 2,3,2-tet > cyclam. This order parallels that found in previous studies in aqueous solution. However, in this study, enthalpic factors were found to be dominant whereas enthalpic and entropic factors have been reported to be comparable in magnitude in aqueous solution. Optical studies from 200–1500 nm were conducted on 0.01 M acetonitrile solutions of the complexes from 25–65 °C. Only small changes in the intensity and position of the optical bands were observed with temperature except for the 2,3,2-tet (468 nm), 3,2,3-tet (450 nm), and cyclam (460 nm) complexes. These indicated bands increase with increasing temperature, which can be explained by assuming a square-planar (diamagnetic)-octahedral (paramagnetic) equilibrium in agreement with the magnetic susceptibility data. Band assignments were made for the cis- and trans-octahedral isomers for each of the complexes. The order of cis-octahedral character for the complexes was found to be 2,2,2- tet, 3,3,3-tet > 2,3,2-tet > 3,2,3-tet > cyclam in agreement with previous studies in aqueous, DMSO, and DMF solutions.     

Microscopy of xanthan/galactomannan mixtures

Polarization microscopy has been used to investigate the structure of 50/50 xanthan/galactomannan (guar gum or locust bean gum) mixtures in aqueous solution, the total concentration ranging from 0.5 to 4%. By the use of polarized light microscopy birefringent areas resulting from the formation of cholesteric mesophases in xanthan gum was clearly seen as has previously been reported by several authors. In xanthan/galactomannan mixtures, we also observed birefringent areas. Moreover, these zones in the blend appeared more anisotropic than with xanthan gum alone. This suggests that xanthan molecules organize themselves as liquid crystalline mesophases in definite enriched xanthan areas resulting from a concentration of xanthan inside these birefringent zones. Upon heating, this anisotropy disappears at a temperature well below the helix-coil transition temperature of xanthan molecules. In fact, this loss of order of the mixed system occurs at the same temperature as the melting temperature of the gel, as assessed by the use of rheological measurements. Since the ordered helical structure of the xanthan molecules still exists beyond the melting temperature while anisotropy disappears, this suggests that the xanthan molecules are no longer concentrated in specific areas but more evenly distributed in the medium. Gel melting would, therefore, be the result of the disappearance of these xanthan enriched areas.     

Studies of NAD(H) conformations in solution: A new method using the PMR spectra of cobalt ion—dinucleotide complexes

In aqueous solutions of NAD(H), there is an equilibrium between two different conformations : a “folded” conformation in which adenine and nicotinamide are staked together and an “unfolded” conformation in which the two rings are without interaction.The folded conformation is the more stable in aqueous solution whereas in organic solution it is the unfolded one.As we have previously shown, the PMR spectra of Co²⁺—NAD(H) complexes may be related with the coenzyme conformation giving suggest to a new method for NAD(H) conformational analysis.The results of this method applied to methanol ²H₂O and dioxane/²H₂O solutions are reported in this paper: they are in good accordance with those of spectrofluorimetric analysis.     

Specific ion effects on the solution conformation of poly-L-proline

The effects of various electrolytes on the conformation of poly-L -proline II in aqueous and nonaqueous solution have been investigated by optical rotatory techniques. It is shown that these agents induce a linear decrease in the corrected specific levorotaton of poly-L -proline with increasing salt concentration, with a molar effectiveness which varies from one salt to another. The salt-induced rotatory changes may be divided into anion and cation components, and it is shown that the major specific affectors are the anions, which increase in effectiveness in reducing the corrected specific levorotation in the following sequence: Cl^? < NO₃^? < Br^? < I^? < ClO₄^? < SCN^?. The inorganic monovalent cations tested (Li⁺, Na⁺, K⁺) are all equally effective in decreasing the specific levorotation. Ca⁺⁺ has a marginally greater effect per mole than the inorganic monovalent cations, while the effectiveness of the ganidinium cation is appreciably less. The tetraalkylammonium cations decrease the specific levorotation more effectively than the inorganic monovalent cations, with the molar effectiveness increasing linearly with total content of methyl plus methylene groups. A similar linear increase with increasing methyl plus methylene content is shown by the aliphatic alcohols, though the effect per mole of CH₂ or CH₃ group is appreciably smaller than that shown by the tetraaklylammonium cations. Salts dissolved in essentially anhydron for mamide are also appreciably effective. Selected viscosity experiments have also been carried out to show that the observed effects on specific levorotation have a structural as well as an optical basis. These results are interpreted in terms of a model which involves binding of anions at the imide nitrogen, and cations at the carbonyl oxygen. It is proposed that this binding induces an increase in the double-bond character; of the peptide bond (and thus a shortening of the bond) which is roughly proportional in the polarizability of the bound anion and that this increase is potentiated by cations which decrease the total dielectric constant (e.g., the tetraalkylammonium series), and reduced by cations presenting competitive local anion binding sites (e.g., the guanidinium ion). We propose further that this shortening of the peptide bond is accompanied by a lengthening of the adjacent bond, thus reducing the steric restraints to rotation about this bond (increasing the accessible range of the angle ψ) sufficiently to induce a progressive non-cooperative collapse of the poly-L -proline II structure. Several lines of evidence are presented to support this interpretation. The various neutral salts are also shown to induce a time-dependent precipitation or “salting-out” of poly-L -proline from solution. In order of decreasing molar effectiveness as salting-out agents in this system, the various ions may be ranked: SO₄^? > Ac^? > Cl^? > Br^? > SCN^? > I^? > ClO₄^?; and K⁺ ? Na⁺ > Li⁺ > Ca⁺⁺. These rankings follow the usual Hofmeister or lyotropic series, and are quite different from hose which apply to the effects on solution conformation of poly-L -proline.     

A vibrational spectroscopic study of the self-association of polyinosinic acid and polyguanylic acid in aqueous solution

We have studied by Raman and ir spectroscopy the structure of self-associated polyinosinic acid and polyguanylic acid in aqueous solution. The results are consistent with the formation of a four-stranded complex, which melts cooperatively near 60°C in the case of poly (I) in the presence of K⁺ ions. The conformation of the ribose in both systems is mixed C2′-endo/C3′-endo, giving a structure that is intermediate between the extremes proposed previously from x-ray diffraction studies. Characteristic Raman bands for the C2′-endo ribose conformation in polyribonucleotides are identified. The four-stranded structure of poly (I) appears to be very flexible, with ≈15% of the tetrameric segments being disrupted and ≈30% of the ribose units adopting a disordered conformation prior to melting. This disordering process increases to ≈75% above the melting transition, with the remaining ≈25% of the ribose units keeping an ordered C2′-endo or C3′-endo conformation. © 1994 John Wiley & Sons, Inc.