David A. Brant

The sequence statistics and solution conformation of a barley (1→3, 1→)-β-d-glucan

Abstract The sequence statistics and aqueous solution conformation of the 40° water-soluble (1→3,1→)-β- d -glucan isolated from barley ( Hordeum vulgare ) have been modeled realistically using the known sequence-distributions of (1→3) and (1→4) linkages, theoretical conformational analysis, and the statistical mechanical theory of polymer-chain conformation. This barley β-glucan fraction consists of (1→4)-β-glucooligosaccharides, predominantly of d.p. 4 or less, joined by single β-(1→3) linkages. Approximate treatments fo the sequence statistics which do not take into account the small mole fraction (∼2%) of (1→4)-β-glucooligosaccharides of d.p. ∼10 significantly underestimate the chain extension in solution. A correct prediction of the observed chain extension is achieved when these longer, highly extended (1→4)-β-glucooligosaccharide blocks are included in a model which randomly incorporates all (1→4)-β-glucooligosaccharide segments in the proportions observed experimentally. Chain flexibility in the 40° water-soluble β-glucan fraction is shown to arise principally from the siolated β-(1→3) linkages; blocks of two or more continguous β-(1→3) linkages provide a source of additional flexibility which may influence the properties of barley β-glucan fractions containing a significant proportion of such sequences.

Aggregates in acidic solutions of chitosans detected by static laser light scattering

Abstract Chitosans having degrees of N -acetylation, F A , ranging from 0 to 0·6, were randomly degraded to different molecular weights and studied by multi angle static laser light scattering (LLS). Under the given experimental conditions, negative second virial coefficients of the solutions, A ′ 2 , revealed the presence of concentration dependent aggregates. Attempts to remove the aggregates, or to influence the aggregation behavior, were made by ultracentrifugation and extensive filtering of the solutions. Modification of the solvent conditions such as pH, ionic strength and temperature were carried out, and chitosan solutions were digested with an acidic proteinase. Non-degraded samples and chitosans prepared by both heterogeneous and homogeneous N -deacetylation of chitin were also studied. In all cases, the negative A ′ 2 remained. However, it was observed that ultracentrifugation and filtering of the solutions decreased the measured molecular weights and radii of gyration, indicating that some of the material of high molecular weight and size could be removed by ultracentrifugation and filtration. The chemical nature of the physical basis of the molecular association was not revealed. Nevertheless, by the use of gel permeation chromatography coupled to an on-line low angle laser light scattering instrument and a differential refractive index concentration detector (HPSEC-LALLS-RI), a bimodal molecular weight distribution was observed in which about 5% of the sample had a very high molecular weight. These results coupled with the positive virial coefficients obtained earlier from osmotic pressure measurements suggest that a small fraction of the chitosan is aggregated to high molecular weight material, probably following a closed association model. Electron microscopy revealed the presence of some supramolecular structures. The positive second virial coefficients obtained earlier from osmometry are in harmony with these findings. The results demonstrate the occurrence of reversible aggregation in chitosan solutions. Static laser light scattering therefore cannot readily be used to determine molecular weights and sizes of chitosans under these conditions. It was not possible to correlate the extent of aggregation with the chemical composition of the chitosans.

Imaging of individual biopolymers and supramolecular assemblies using noncontact atomic force microscopy.

A variety of biopolymers is imaged using noncontact atomic force microscopy. Samples are prepared by aerosol spray deposition of aqueous solutions on freshly cleaved mica followed by air drying. The distributions of contour lengths and chain or fibril thicknesses normal to the mica substrate can be measured for individual polymer molecules or molecular assemblies. In many cases it is possible to conclude that the structures imaged and quantitatively analyzed are representative of those present in solution and not artifacts of the deposition/dessication process. Imaging of linear and cyclic triple helices of the polysaccharide scleroglucan is demonstrated. Measurements of the triple helix thickness normal to the mica surface are analyzed, and successful measurements of the molecular weight distribution and mean molar mass are described. It is demonstrated that the extent of chain association in the polysaccharide xanthan can be modulated by the addition of low molecular weight salts. The contour length and chain thickness distributions in a xanthan fraction are presented. Increases in the extent of chain association with increasing polymer concentration are documented for the gelling polysaccharide gellan, and the formation of stiff fibrillar gellan aggregates in the presence of added low molecular salt is demonstrated. Images are presented of the polysaccharide kappa-carrageenan in its disordered, and presumably single-stranded, state. Biopolymers other than polysaccharides can be imaged by the same technique; this is demonstrated with the fibrous protein collagen. In general it is shown that aerosol spray deposition of biopolymer samples can be used in conjunction with noncontact atomic force microscopy to provide a fast, reliable, and reproducible method for assessing the size and shape distributions of individual biological macromolecules and macromolecular assemblies in solution with a minimum of time and effort devoted to sample preparation.

Imaging of carrageenan macrocycles and amylose using noncontact atomic force microscopy

Samples of kappa-carrageenan, iota-carrageenan, and synthetic amylose have been examined by atomic force microscopy (AFM). All samples were spray deposited from aqueous solutions onto freshly cleaved mica, air dried, and imaged in air using noncontact atomic force microscopy (NCAFM). Images of single stranded amylose and carrageenan are presented. At relatively low polymer concentrations in the presence of NaCl iota-carrageenan formed circles that appear to be predominantly head-to-tail associated unimeric duplex (double stranded) structures. At higher iota-carrageenan concentrations the polymer forms circles and aggregates that appear to involve dimeric duplex structure. Direct comparison of synthetic amylose molecular weights determined from NCAFM images with results from solution measurements showed that NCAFM provides an excellent way to measure amylose molecular weight and molecular weight distribution. It is shown that synthetic amylose is single stranded in aqueous solution and that the chain length distribution is broader than the Poisson distribution anticipated from polymerization theory.

Light scattering reveals micelle-like aggregation in the (1→3),(1→4)-β-D-glucans from oat aleurone

Abstract Fractionated samples of (1→3),(1→4)-β- D -glucans from oat aleurone (‘oat bran’) with a relatively narrow molecular weight distribution were characterized by total intensity light scattering. Typical Zimm plots showed that for each concentration the angular dependence could be fitted with a straight line. For each angle, however, the concentration dependence showed a distinctive curvature and the negative second virial coefficient typical of a reversibly aggregating system. The shape of the Zimm plots was the same at increased temperature (60°C) in different solvents (1 mol/ dm3 aqueous Lil, 4 mol/dm3 aqueous urea and dimethylsulfoxide) and after ultracentrifugation (300 000 g for 4 h). The extrapolated data at zero concentration and zero angle nevertheless yielded weight average molecular weights (Mw) in the same range as the osmometric number average molecular weights (Mn) determined previously. The relationship between the root-mean-square z-average radius of gyration (Rg) and Mw was found to be Rg (nm), = 0.03 × Mw0.59 in good accord with expectation from polysaccharides of similar structure and consistent with earlier realistic molecular modeling of these β- D -glucans. A cooperative, closed association model is described, and it is shown that the apparent Mw obtained at different concentrations from the light scattering data at zero angle can be closely fitted with the Mw calculated from the model, assuming that multimers consisting of 4–5 unimers are formed. This model predicts—in contrast to experimental observation—a strongly negative osmotic second virial coefficient. A refined aggregation model in which only a fraction of the unimers are assumed capable of involvement in association to form large, cooperatively stabilized aggregates fits both the present light scattering and the earlier osmotic pressure data quite satisfactorily.

Temperature and molecular weight dependence of the unperturbed dimensions of aqueous pullulan

Abstract The intrinsic viscosities, [η], and light scattering molecular weights, M w , second virial coefficients, A′2, and root-mean-square radii of gyration, 〈 S z 2 〉 1 2 , have been measured for a series of pullulan fractions in aqueous solution at 25°C. These have been used to establish the dependence on molecular weight of [η], A′2, and 〈S z 2 〉 1 2 and to deduce the limiting characteristic ratio of the unperturbed mean-square end-to-end distance C∞ (4.3−0.2) for aqueous pullulan at this temperature. The temperature dependence of C∞ was determined from measurements of the same properties for selected fractions at a series of temperatures, and the temperature coefficient dln C∞/dT (−0.0043 deg−1) was established.

Novel approaches to the analysis of polysaccharide structures

Recently, atomic force microscopy has been used to image a variety of polysaccharides and map their distribution on cell surfaces. The mechanical response of polysaccharides to tensile stress has been investigated in single-molecule force spectroscopy experiments. Small-angle X-ray scattering has provided a probe of polysaccharide structure operating in a size range (2-25 nm) that is intermediate between those accessible using nuclear magnetic resonance and light scattering.

Kelco microbial polysaccharides S-130 (welan) and S-657 Display similar dilute aqueous solution behavior

The comb-like, branched microbial polysaccharides S-130 (welan gum) from Alcaligenes ATCC 31555 and S-657 from Xanthomonas ATCC 53159, produced by the Kelco Division of Merck, have potential commercial applications as non-gelling water-soluble thickening and suspending agents. Each is structurally related to the Kelco linear copolysaccharide S-60 (gellan gum) from Auromonas ATCC 31461 which forms gels in aqueous salt solutions and has the repeating unit structure [→ 3)-β-d-Glcp-(1 → 4)-β-d-GlcpA-(1 → 4)-β-d-Glcp- (1 → 4)-α-l-Rhap (1 → ]. In S-130 this backbone is regularly glycosylated at C(3) of the second β-d-Glcp with α-l-Rhap (67%) or α-l-Manp (33%), while in S-657 regular glycosylation occurs at the same backbone site with α-l-Rhap-(1 → 4)-α-l-Rhap. Both S-130 and S-657 were purified, degraded to various extents by sonication and, for most samples, fractionally precipitated into moderately dispersed fractions differing in mean molecular weight. Potentiometric proton titration and investigations of the dependence of intrinsic viscosity on ionic strength suggest only very weak polyelectrolyte behavior. For neither polymer does the temperature or ionic strength dependence of optical activity or reduced specific viscosity present evidence of a conformational change in the temperature range 20≤T≤62°C. Light scattering as a function of ionic strength discloses a modest tendency of both polymers to aggregate with increasing aqueous NaCl concentration in the range 0·001–0·100 m; intrinsic viscosities were large relative to the molecular weights and essentially constant over the same range of salt concentration. The mean chain extension per back-bone sugar residue measured by light scattering, as well as the molecular weight dependence of the intrinsic viscosity, suggest a highly extended chain configuration, similar for the two polymers and comparable to that of the most highly extended cellulose derivatives. This is found despite the periodic occurrence in S-130 and S-657 of the α-(1 → 3)-linkage, which is expected on elementary grounds to disrupt the essentially rectilinear propagation of the otherwise cellulose-like backbone and generate a less extended chain configuration.

Thermal treatment of semi-dilute aqueous xanthan solutions yields weak gels with properties resembling hyaluronic acid

Semi-dilute (ca 2 g/dl) aqueous xanthan (mean molar mass ca 1 x 10(6) g/mol), when heated in the presence of 0.1 M NaCl to a temperature above the order<-->disorder transition temperature, forms highly viscoelastic solutions when returned to room temperature. The steady shear and dynamic rheological behaviour of these solutions discloses a weak gel structure, the viscosity of which is unusually sensitive to the rate of shear. In shear thinning behaviour these heat and salt treated xanthan solutions mimic the properties of the aqueous hyaluronic acid solutions widely used in viscosurgical techniques. The double stranded model of native xanthan is invoked to interpret the observed behaviour of heat and salt treated semi-dilute aqueous xanthan.

Theoretical interpretation of the unperturbed aqueous solution configuration of pullulan

Abstract A structurally realistic theoretical model for the pullulan chain is developed and refined to be consistent with the experimentally measured unperturbed dimensions of aqueous pullulan and with the temperature dependence of the unperturbed dimensions. The model is based on the mean structural geometry for an α- D -glucose residue recommended by Arnott and Scott and incorporates, in estimates of the conformational energy of the chain, a treatment of the gauche , or exo -anomeric, effect proposed by Abe and Mark. The refined chain model is used to generate computer drawn images of the pullulan chain in conformations typical of those characterizing the pullulan random coil in aqueous solution. The propensity of the pullulan chain trajectory to persist in its original direction is described in terms of an appropriately defined residue vector correlation function. The conformational energy functions associated with the refined chain model suggest, in contrast to some other recent treatments, that the trans-gauche rotational isomeric state of the C(5)—C(6) bond contributes to the configurational properties of oligo- and polysaccharides containing the α- D -glucopyranosyl-(1 → 6)-α- D -glucopyranosyl moiety.