Global hydrodynamic analysis of the molecular flexibility of galactomannans

In the past, intrinsic viscosity and sedimentation velocity analyses have been used separately to assess the conformation and flexibility of guar and locust bean gum galactomannans based on worm-like chain and semi-flexible coil models. Publication of a new global method combining data sets of both intrinsic viscosity and sedimentation coefficient with molecular weight, and minimising a target (error) function now permits a more robust analysis. Using this approach, values for the persistence length of (10 ± 2) nm for guar and (7 ± 1) nm for locust bean gum are returned if the mass per unit length ML is floated as a variable. Using a fixed mass per unit length based on the known compositional data of each galactomannan yields a similar value for Lp in both cases, (8 ± 1) nm for guar and (9 ± 1) nm for locust bean gum, with combined set of data yielding (9 ± 1) nm: within experimental error the flexibilities of both galactomannans are very similar. © 2007 Elsevier Ltd. All rights reserved

Molecular flexibility of citrus pectins by combined sedimentation and viscosity analysis

The flexibility/rigidity of pectins plays an important part in their structure-function relationship and therefore on their commercial applications in the food and biomedical industries. Earlier studies based on sedimentation analysis in the ultracentrifuge have focused on molecular weight distributions and qualitative and semi-quantitative descriptions based on power law and Wales-van Holde treatments of conformation in terms of "extended" conformations [Harding, S. E., Berth, G., Ball, A., Mitchell, J.R., & Garcìa de la Torre, J. (1991). The molecular weight distribution and conformation of citrus pectins in solution studied by hydrodynamics. Carbohydrate Polymers, 168, 1-15; Morris, G. A., Foster, T. J., & Harding, S.E. (2000). The effect of degree of esterification on the hydrodynamic properties of citrus pectin. Food Hydrocolloids, 14, 227-235]. In the present study, four pectins of low degree of esterification 17-27% and one of high degree of esterification (70%) were characterised in aqueous solution (0.1 M NaCl) in terms of intrinsic viscosity [η], sedimentation coefficient (s°20,w) and weight average molar mass (Mw). Solution conformation/flexibility was estimated qualitatively using the conformation zoning method [Pavlov, G.M., Rowe, A.J., & Harding, S.E. (1997). Conformation zoning of large molecules using the analytical ultracentrifuge. Trends in Analytical Chemistry, 16, 401-405] and quantitatively (persistence length Lp) using the traditional Bohdanecky and Yamakawa-Fujii relations combined together by minimisation of a target function. Sedimentation conformation zoning showed an extended coil (Type C) conformation and persistence lengths all within the range Lp=10-13 nm (for a fixed mass per unit length)

Polyvinyl chloride insoluble gels: Their structure and effect on properties of cast films

The effect of insoluble gel material, crystalline aggregates, and suspension particle membranes on the physical and mechanical properties of solution cast films of suspension PVC has been studied. The chemical structure and morphology of the insoluble gel material was also studied. The suspension particle membranes are found to contain significant amounts of hydroxyl and carbonyl groups but are still predominately PVC. These particles affect the transparency of cast films as well as the surface topography by forming discrete clusters upon drying which are uniformly distributed throughout the films. Finally, insoluble gel material is found to have no significant effect on the low and high speed mechanical properties of the films.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38100/1/760140508_ftp.pd

Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates – in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium “SEDFIT MSTAR” analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), “Conformation Zoning” and Wales-van Holde approaches showed a high degree of flexibility – at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein

A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate

Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 106 g.mol−1) compared to the native (Mw ~ 1.2 × 106 g.mol−1). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 106 g.mol−1), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution

Central effects of atropine upon aversive classical conditioning in rabbits

Rabbits were given classical discrimination conditioning with one of two tones followed by shock. In Experiment I, 40 rabbits were trained under saline, 10, 18 or 26 mg/kg atropine sulfate or 18 mg/kg methylatropine. Six rabbits in Experiment 2 were conditioned, then given further sessions with saline, and 18, 26 and 34 mg/kg atropine sulfate and methylatropine. In Experiment 3, 18 rabbits were conditioned and then given two extinction sessions under saline or 34 mg/kg atropine sulfate or methylatropine followed by extinction under saline. Chief findings were (a) atropine sulfate but not methylatropine disrupted acquisition and maintenance of conditioned eyeblinks, (b) neither drug affected unconditioned blinks, (c) fewer blinks occurred in extinction under atropine sulfate than under methylatropine or saline, (d) rabbits extinguished under atropine sulphate showed higher percentages of eyeblinks when tested without drug. Disruptions in performance of learned eyeblink responses appeared to be due to drug interference with central cholinergic transmission.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46381/1/213_2004_Article_BF00406735.pd

Application of recent advances in hydrodynamic methods for characterising mucins in solution

Mucins are the primary macromolecular component of mucus—nature’s natural lubricant—although they are poorly characterised heterogeneous substances. Recent advances in hydrodynamic methodology now offer the opportunity for gaining a better understanding of their solution properties. In this study a combination of such methods was used to provide increased understanding of a preparation of porcine intestinal mucin (PIM), MUC2 mucin, in terms of both heterogeneity and quantification of conformational flexibility. The new sedimentation equilibrium algorithm SEDFIT-MSTAR is applied to yield a weight average (over the whole distribution) molar mass of 7.1 × 106 g mol−1, in complete agreement with size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), which yielded a value of 7.2 × 106 g mol−1. Sedimentation velocity profiles show mucin to be very polydisperse, with a broad molar mass distribution obtained using the Extended Fujita algorithm, consistent with the elution profiles from SEC-MALS. On-line differential pressure viscometry coupled to the SEC-MALS was used to obtain the intrinsic viscosity [η] as a function of molar mass. These data combined with sedimentation coefficient data into the global conformation algorithm HYDFIT show that PIM has a flexible linear structure, with persistence length Lp ~10 nm and mass per unit length, ML ~2380 g mol−1 nm−1, consistent with a Wales-van Holde ratio of ~1.2 obtained from the concentration dependence of the sedimentation coefficient

Ultracentrifuge methods for the analysis of polysaccharides, glycoconjugates, and lignins

Although like proteins, polysaccharides are synthesized by enzymes, unlike proteins there is no template. This means that they are polydisperse, do not generally have compact folded structures, and are often very large with greater nonideality behavior in solution. This chapter considers the relevant analytical ultracentrifuge methodology available for characterizing these and related carbohydrate-based systems and information this methodology supplies, in terms of sizes, shapes, and interactions using a comprehensive range of examples, including glycoconjugates and lignins. The relevance and potential of recent software developments such as SEDFIT-MSTAR, the Extended Fujita algorithm, and HYDFIT are considered