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

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

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

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

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

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

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

Effectiveness of proactive telephone counselling for smoking cessation in parents: Study protocol of a randomized controlled trial

Contains fulltext : 99284.pdf (publisher's version ) (Open Access)Background Smoking is the world's fourth most common risk factor for disease, the leading preventable cause of death, and it is associated with tremendous social costs. In the Netherlands, the smoking prevalence rate is high. A total of 27.7% of the population over age 15 years smokes. In addition to the direct advantages of smoking cessation for the smoker, parents who quit smoking may also decrease their children's risk of smoking initiation. Methods/Design A randomized controlled trial will be conducted to evaluate the effectiveness of proactive telephone counselling to increase smoking cessation rates among smoking parents. A total of 512 smoking parents will be proactively recruited through their children's primary schools and randomly assigned to either proactive telephone counselling or a control condition. Proactive telephone counselling will consist of up to seven counsellor-initiated telephone calls (based on cognitive-behavioural skill building and Motivational Interviewing), distributed over a period of three months. Three supplementary brochures will also be provided. In the control condition, parents will receive a standard brochure to aid smoking cessation. Assessments will take place at baseline, three months after start of the intervention (post-measurement), and twelve months after start of the intervention (follow-up measurement). Primary outcome measures will include sustained abstinence between post-measurement and follow-up measurement and 7-day point prevalence abstinence and 24-hours point prevalence abstinence at both post- and follow-up measurement. Several secondary outcome measures will also be included (e.g., smoking intensity, smoking policies at home). In addition, we will evaluate smoking-related cognitions (e.g., attitudes towards smoking, social norms, self-efficacy, intention to smoke) in 9-12 year old children of smoking parents. Discussion This study protocol describes the design of a randomized controlled trial to evaluate the effectiveness of proactive telephone counselling in smoking cessation. It is expected that, in the telephone counseling condition, parental smoking cessation rates will be higher and children's cognitions will be less favorable about smoking compared to the control condition. Trial registration The protocol for this study is registered with the Netherlands Trial Register NTR2707.6 p

Influence of family and friend smoking on intentions to smoke and smoking-related attitudes and refusal self-efficacy among 9-10 year old children from deprived neighbourhoods: a cross-sectional study.

BACKGROUND: Smoking often starts in early adolescence and addiction can occur rapidly. For effective smoking prevention there is a need to identify at risk groups of preadolescent children and whether gender-specific intervention components are necessary. This study aimed to examine associations between mother, father, sibling and friend smoking and cognitive vulnerability to smoking among preadolescent children living in deprived neighbourhoods. METHODS: Cross-sectional data was collected from 9-10 year old children (n =1143; 50.7% girls; 85.6% White British) from 43 primary schools in Merseyside, England. Children completed a questionnaire that assessed their smoking-related behaviour, intentions, attitudes, and refusal self-efficacy, as well as parent, sibling and friend smoking. Data for boys and girls were analysed separately using multilevel linear and logistic regression models, adjusting for individual cognitions and school and deprivation level. RESULTS: Compared to girls, boys had lower non-smoking intentions (P = 0.02), refusal self-efficacy (P = 0.04) and were less likely to agree that smoking is 'definitely' bad for health (P < 0.01). Friend smoking was negatively associated with non-smoking intentions in girls (P < 0.01) and boys (P < 0.01), and with refusal self-efficacy in girls (P < 0.01). Sibling smoking was negatively associated with non-smoking intentions in girls (P < 0.01) but a positive association was found in boys (P = 0.02). Boys who had a smoking friend were less likely to 'definitely' believe that the smoke from other people's cigarettes is harmful (OR 0.57, 95% CI: 0.35 to 0.91, P = 0.02). Further, boys with a smoking friend (OR 0.38, 95% CI: 0.21 to 0.69, P < 0.01) or a smoking sibling (OR 0.45, 95% CI: 0.21 to 0.98) were less likely to 'definitely' believe that smoking is bad for health. CONCLUSION: This study indicates that sibling and friend smoking may represent important influences on 9-10 year old children's cognitive vulnerability toward smoking. Whilst some differential findings by gender were observed, these may not be sufficient to warrant separate prevention interventions. However, further research is needed