Aspects of the analytical ultracentrifuge determination of the molar mass distribution of polysaccharides

Molar mass or ‘molecular weight’ is one of the most fundamental parameters describing a macromolecule. Because of their polydisperse nature, polysaccharides are usually described by distributions of molar mass. SEC-MALS (size exclusion chromatography coupled to multi-angle light scattering) is often a convenient method of choice, but there are many instances where it is unsuitable. Modern AUC (analytical ultracentrifuge) methods provide a valuable alternative – now easier to use than before – and, after briefly reviewing some older procedures, we highlight two recently published and complementary methods, namely, the ‘Extended Fujita’ approach for the analysis of sedimentation velocity data and SEDFIT-MSTAR for the analysis of sedimentation equilibrium data. Nonideality needs to be considered and can be dealt with in a standard way. These methods can also indicate if associative phenomena are present, which can then be quantified using more complex AUC algorithms

Controlled depolymerisation, as assessed by analytical ultracentrifugation, of low molecular weight chitosan for potential use in archaeological conservation

The heterogeneity and molecular weight of a chitosan of low molecular weight (molar mass) and low degree of acetylation (0.1), for potential use as a consolidant for decayed archaeological wood, has been examined by sedimentation velocity and sedimentation equilibriumin the analytical ultracentrifuge before and after depolymerisation. Sedimentation velocity before polymerisation revealed a uniform distribution of sedimentation coefficient with little concentration dependence. SEDFIT-MSTAR analysis revealed a weight average molecular weight Mw of (14.2 + 1.2) kDa, and polydispersity index of ~ 1.2. Further analysis using MULTISIG revealed a distribution of material between 2-20 kDa and consistent with the weight average Mw. Controlled depolymerisation using hydrogen peroxide and UV in an acetic acid medium reduced this to (4.9 + 0.7) kDa, with a similar polydispersity. The depolymerised material appears to be within the range that has been predicted to fully penetrate into archaeological wood. The consequences for this and the use of the analytical ultracentrifuge in wood conservation strategies is considered

Aminoethyl substitution enhances the self-assembly properties of an aminocellulose as a potential archaeological wood consolidant

The 6-deoxy-6-aminocelluloses—or “aminocelluloses”—are a class of synthetic natural cellulose derivatives which are mostly aqueous soluble and have excellent film-forming properties. Recent studies have connected these properties at the molecular level with protein-like self-associative behaviour for a range of aminocelluloses including a 6-deoxy-6-(ω-aminoethyl) aminocellulose AEA-1 with the association being a two-stage process—a reversible oligomerisation followed by further (semi-reversible) aggregation into larger structures. Here, we synthesise and compare a new 6-deoxy-6-(ω-aminoethyl) aminocellulose AEA-1′ with different degree of substitution with one with further alkyl derivatisation, namely 6-deoxy-6-(ω-hydroxyethyl) aminocellulose HEA-1′. As with AEA-1, sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge still show a two-stage process for both AEA-1′ and HEA-1′, with the latter giving higher molar masses. The consequences of these properties for use as consolidants for archaeological wood are considered

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

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 in the analytical ultracentrifugation provides a powerful method with no supplementary immobilization, columns or membranes required. It is particularly powerful when used in conjunction with its sister technique, namely sedimentation velocity analysis. We describe key approaches now available and their application to the characterisation 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 DonWinzor over several decades of research

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-(v-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

Isolation and Biophysical Characterisation of Bioactive Polysaccharides from Cucurbita Moschata (Butternut Squash)

Cucurbits are plants that have been used frequently as functional foods. This study includes the extraction, isolation, and characterisation of the mesocarp polysaccharide of Cucurbita moschata. The polysaccharide component was purified by gel filtration into three fractions (NJBTF1, NJBTF2, and NJBTF3) of different molecular weights. Characterisation includes the hydrodynamic properties, identification of monosaccharide composition, and bioactivity. Sedimentation velocity also indicated the presence of small amounts of additional discrete higher molecular weight components even after fractionation. Sedimentation equilibrium revealed respective weight average molecular weights of 90, 31, and 19 kDa, with the higher fractions (NJBTF1 and NJBTF2) indicating a tendency to self-associate. Based on the limited amount of data (combinations of 3 sets of viscosity and sedimentation data corresponding to the 3 fractions), HYDFIT indicates an extended, semi-flexible coil conformation. Of all the fractions obtained, NJBTF1 showed the highest bioactivity. All fractions contained galacturonic acid and variable amounts of neutral sugars. To probe further, the extent of glycosidic linkages in NJBTF1 was estimated using gas chromatography–mass spectrometry (GCMS), yielding a high galacturonic acid content (for pectin polysaccharide) and the presence of fructans—the first evidence of fructans (levan) in the mesocarp. Our understanding of the size and structural flexibility together with the high bioactivity suggests that the polysaccharide obtained from C. moschata has the potential to be developed into a therapeutic agent

Glargine and degludec: solution behaviour of higher dose synthetic insulins

Single, double and triple doses of the synthetic insulins glargine and degludec currently used in patient therapy are characterised using macromolecular hydrodynamic techniques (dynamic light scattering and analytical ultracentrifugation) in an attempt to provide the basis for improved personalised insulin profiling in patients with diabetes. Using dynamic light scattering and sedimentation velocity in the analytical ultracentrifuge glargine was shown to be primarily dimeric under solvent conditions used in current formulations whereas degludec behaved as a dihexamer with evidence of further association of the hexamers (“multi-hexamerisation”). Further analysis by sedimentation equilibrium showed that degludec exhibited reversible interaction between mono- and-di-hexamer forms. Unlike glargine, degludec showed strong thermodynamic non-ideality, but this was suppressed by the addition of salt. With such large injectable doses of synthetic insulins remaining in the physiological system for extended periods of time, in some case 24–40 hours, double and triple dose insulins may impact adversely on personalised insulin profiling in patients with diabetes

MultiSig: a new high-precision approach to the analysis of complex biomolecular systems

MultiSig is a newly developed mode of analysis of sedimentation equilibrium (SE) experiments in the analytical ultracentrifuge, having the capability of taking advantage of the remarkable precision (~0.1 % of signal) of the principal optical (fringe) system employed, thus supplanting existing methods of analysis through reducing the ‘noise’ level of certain important parameter estimates by up to orders of magnitude. Long-known limitations of the SE method, arising from lack of knowledge of the true fringe number in fringe optics and from the use of unstable numerical algorithms such as numerical differentiation, have been transcended. An approach to data analysis, akin to ‘spatial filtering’, has been developed, and shown by both simulation and practical application to be a powerful aid to the precision with which near-monodisperse systems can be analysed, potentially yielding information on protein-solvent interaction. For oligo- and poly-disperse systems the information returned includes precise average mass distributions over both cell radial and concentration ranges and mass-frequency histograms at fixed radial positions. The application of MultiSig analysis to various complex heterogenous systems and potentially multiply-interacting carbohydrate oligomers is described

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