Richard B. Gillis

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.

Protein-like fully reversible tetramerisation and super-association of an aminocellulose

Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered “protein-like” and what might be considered as “carbohydrate-like” behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for interactions with vancomycin

VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARA two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSA sensory component has not hitherto been identified. Here we employ purified, intact and active VanSA membrane protein (henceforth referred to as VanS) in analytical ultracentrifugation experiments to study VanS oligomeric state and conformation in the absence and presence of vancomycin. A combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge (SEDFIT, SEDFIT-MSTAR and MULTISIG analysis) showed that VanS in the absence of the ligand is almost entirely monomeric (molar mass M = 45.7 kDa) in dilute aqueous solution with a trace amount of high molar mass material (M ~ 200 kDa). The sedimentation coefficient s suggests the monomer adopts an extended conformation in aqueous solution with an equivalent aspect ratio of ~(12 ± 2). In the presence of vancomycin over a 33% increase in the sedimentation coefficient is observed with the appearance of additional higher s components, demonstrating an interaction, an observation consistent with our circular dichroism measurements. The two possible causes of this increase in s – either a ligand induced dimerization and/or compaction of the monomer are considered.

Application and use of Inulin as a tool for therapeutic drug delivery.

Abstract Inulin is a polysaccharide with an extensive range of therapeutic uses such as a vehicle in drug delivery vehicle, as a diagnostic/analytical tool or as a dietary fibre with additional health benefits. In the main, much research has focussed on inulin as a drug delivery carrier for colon-targeted drug delivery. The justification for this is its potential to survive the stomachs acidic environment. This unique stability and strength is utilized in many ways to deliver drugs safely to colon, where they can be easily absorbed through the gut epithelium into the blood. Inulin based hydrodynamic research will be useful to discover the potential of inulin.

Synthesis and characterisation of lignin-like oligomers as a bio-inspired consolidant for waterlogged archaeological wood

Abstract The development of new materials for the consolidation of waterlogged archaeological wood from sustainable sources is an important area of research, as the most widely used consolidant today is petroleum based. Ideally a new consolidant will interact with the existing wood structure, ensuring maximum compatibility. Lignin is often the major component remaining in archaeological wood, as it is less susceptible to degradation than holocellulose. Therefore, in order to maximise the potential for interaction with the wood cells, lignin-like oligomers have been synthesized from isoeugenol using a water soluble copper salen catalyst at pH 10, giving a weight average Mw of 1.6 kDa. Analysis by NMR spectroscopy has shown that the oligomers have a lignin-like structure with β-O-4′, β-β′ and β-5′ connections. A 10 w/w% solution of the oligomers in ethyl acetate was found to thoroughly penetrate 1 cm3 samples of waterlogged archaeological wood (density of 0.146 g/mL, maximum water content of 620%) after 14 days impregnation, as determined by FTIR spectroscopy. No impregnation material could be seen by SEM, suggesting that it coats the cell walls upon drying. This indicates that dehydrogenated polymers penetrate waterlogged archaeological wood well and have the potential to be developed into consolidants.

Full hydrodynamic reversibility of the weak dimerization of vancomycin and elucidation of its interaction with VanS monomers at clinical concentration

The reversibility and strength of the previously established dimerization of the important glycopeptide antibiotic vancomycin in four different aqueous solvents (including a medically-used formulation) have been studied using short-column sedimentation equilibrium in the analytical ultracentrifuge and model-independent SEDFIT-MSTAR analysis across a range of loading concentrations. The change in the weight average molar mass Mw with loading concentration was consistent with a monomer-dimer equilibrium. Overlap of data sets of point weight average molar masses Mw(r) versus local concentration c(r) for different loading concentrations demonstrated a completely reversible equilibrium process. At the clinical infusion concentration of 5 mg.mL−1 all glycopeptide is dimerized whilst at 19 µg.mL−1 (a clinical target trough serum concentration), vancomycin was mainly monomeric (<20% dimerized). Analysis of the variation of Mw with loading concentration revealed dissociation constants in the range 25-75 μM, commensurate with a relatively weak association. The effect of two-fold vancomycin (19 µg.mL−1) appears to have no effect on the monomeric enterococcal VanS kinase involved in glycopeptide resistance regulation. Therefore, the 30% increase in sedimentation coefficient of VanS on adding vancomycin observed previously is more likely to be due to a ligand-induced conformational change of VanS to a more compact form rather than a ligand-induced dimerization.

A review of modern approaches to the hydrodynamic characterisation of polydisperse macromolecular systems in biotechnology.

This short review considers the range of modern techniques for the hydrodynamic characterisation of macromolecules – particularly large glycosylated systems used in the food, biopharma and healthcare industries. The range or polydispersity of molecular weights and conformations presents special challenges compared to proteins. The review is aimed, without going into any great theoretical or methodological depth, to help the Industrial Biotechnologist choose the appropriate methodology or combination of methodologies for providing the detail he/she needs for particular applications.

The Effectiveness of Lifestyle Adaptation for the Prevention of Prediabetes in Adults: A Systematic Review

Diabetes prevalence is increasing exceptionally worldwide and with this come associated healthcare costs. The primary outcome of this systematic review was to assess glycaemic control and incidence of Type 2 diabetes mellitus (T2DM) diagnosis after exercise and dietary intervention (measured with any validated scale). The secondary outcome assessed body mass index change, weight change, and physical exercise capacity after diet and exercise intervention (measured with any validated scale). 1,780 studies were identified from searching electronic databases. Relevant studies went through a selection process. The inclusion criteria for all studies were people with prediabetes diagnosed by either impaired glucose tolerance (IGT) or impaired fasting glucose (IFG). Lifestyle adaptation reduced the incidence of diabetes development more than standard treatment. Furthermore, better glycaemic control, improved physical exercise capacity, and increased weight reduction were observed with lifestyle intervention over standard treatment. Finally, improvements over the long term deteriorated, highlighting problems with long-term adherence to lifestyle changes. Overall, cumulative incidence of diabetes is drastically reduced in the intervention groups compared to control groups (standard care). Furthermore, glycaemic control was improved in the short term, with many participants reverting to normoglycaemia.

Matrix-free hydrodynamic study on the size distribution and conformation of three technical lignins from wood and non-wood

Abstract Molecular weight (MW) and related conformational data of three commercially available technical lignins (Alcell L, kraft L, and soda L) have been studied by means of analytical ultracentrifugation, taking advantage of some recent developments in both sedimentation velocity and sedimentation equilibrium determinations. The lignins were dissolved in dimethyl sulphoxide (with ca. 90% solubility), and solutions were studied with regards to their oligomeric state, heterogeneity profiles (distribution of sedimentation coefficients), and molecular weight distributions (MWD). Alcell L and soda L have similar properties showing one major low MW component and two minor high MW components, whereas kraft L appears to be larger and more uniform, i.e., it shows a more monodisperse MWD. Weight average molecular weight (Mw) data from sedimentation equilibrium obtained by the new SEDFIT-MSTAR procedure in conjunction with MULTISIG analysis were found to be ~18 kDa (Alcell L), 25 kDa (kraft L), and 15 kDa (soda L). Further analysis of the data by means of the routines MULTISIG and M_INVEQ confirmed the presence of additional components in Alcell L and soda L, and the larger size and high degree of monodispersity of kraft L. The intrinsic viscosity data of the three lignins were found to be very similar in the range of 22–24 ml g-1, and all data were consistent with an elongated plate shape molecular structure with an equivalent discoid aspect ratio ~30.

Assessing sedimentation equilibrium profiles in analytical ultracentrifugation experiments on macromolecules: from simple average molecular weight analysis to molecular weight distribution and interaction analysis.

Molecular weights (molar masses), molecular weight distributions, dissociation constants and other interaction parameters are fundamental characteristics of proteins, nucleic acids, polysaccharides and glycoconjugates in solution. Sedimentation equilibrium analytical ultracentrifugation provides a powerful method with no supplementary immobilization, columns or membranes required. It is a particularly powerful tool when used in conjunction with its sister technique, namely sedimentation velocity. Here, we describe key approaches now available and their application to the characterization of antibodies, polysaccharides and glycoconjugates. We indicate how major complications, such as thermodynamic non-ideality, can now be routinely dealt with, thanks to a great extent to the extensive contribution of Professor Don Winzor over several decades of research.