Challenges for the modern analytical ultracentrifuge analysis of polysaccharides

Harding, Stephen E.

doi:10.1016/j.carres.2005.01.027

Cited by 41 publications

(19 citation statements)

References 88 publications

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“…The modified long (20.0 mm) optical path length double-sector titanium cells with sapphire windows were loaded with 0.15 mL of dialysed sample and a matching amount of reference buffer dialysate in appropriate channels. Samples were centrifuged at rotor speeds selected to give a sufficient fringe increment from meniscus to base50 i.e. 4000, 9000, 9000 and 2000 rpm for native, micro-fluidized, activated polysaccharides and glycoconjugates, respectively.…”

Section: Methodsmentioning

confidence: 99%

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

Morris

Almutairi

Adams

et al. 2016

Sci Rep

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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.

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Section: Methodsmentioning

confidence: 99%

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

Morris

Almutairi

Adams

et al. 2016

Sci Rep

Self Cite

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“…This allows routinely to fit SV data to within the level of the noise of data acquisition, and thereby to extract information with exquisite resolution and sensitivity. Many new applications utilize these techniques, including the characterization of the stoichiometry, thermodynamic, and kinetic parameters of (multi‐)protein interactions,2, 3, 13 the hydrodynamic characterization of the low resolution structure of biomacromolecules and their complexes,14, 15 the size‐distribution of proteins samples,11, 12 membrane proteins,16, 17 carbohydrates,18, 19 the determination of trace aggregate levels in protein therapeutics,6, 20, 21 as well as systems in supramolecular chemistry, biomaterials, and nanoparticles 22–24. For recent reviews, see ref 25–27…”

Section: Introductionmentioning

confidence: 99%

Accounting for Solvent Signal Offsets in the Analysis of Interferometric Sedimentation Velocity Data

Zhao

Brown

Balbo

et al. 2010

Macromolecular Bioscience

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Sedimentation velocity (SV) analytical ultracentrifugation has re-emerged as an important tool in the characterization of biological macromolecules and nanoparticles. The computational analysis of the evolution of the macromolecular concentration profile allows the characterization of many hydrodynamic and thermodynamic properties of the macromolecules and their interactions. The Rayleigh interference optical system is often the detection method of choice, for its usually superior data quality and the wide applicability of refractive index sensitive detection. However, the interference optical system is also sensitive to the redistribution of co-solvent molecules, which are not of primary experimental interest. In principle, their contribution can be eliminated by an exact geometric and compositional match of the sample solution and the reference solution, achieving the complete optical subtraction of unwanted buffer signals. Unfortunately, in practice, this can often not be perfectly achieved for various reasons, leading to signal offsets arising from unmatched sedimentation of solvent components. If unrecognized, this can lead to significant misfit, accompanied by significant errors in the macromolecular sedimentation parameters. In the present work, we describe an approach of computationally accounting for signals from sedimenting buffer components through explicitly modeling their redistribution with Lamm equation solutions, implemented in the software SEDFIT. We demonstrate how this can restore the SV analysis to yield a high quality fit of the data and to provide correct macromolecular sedimentation parameters.

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“…For recent reviews, book chapters, or books, the reader can refer to 11 ,13 ,19 , and 20 . However, in 1992, the Beckman Coulter XLA analytical ultracentrifuge with absorbance optics and then the Beckman Coulter XLI with both absorbance and interference optical systems were commercialized.…”

Section: Introductionmentioning

confidence: 99%

Analytical Ultracentrifugation, a Useful Tool to Probe Intrinsically Disordered Proteins

Manon

Ebel

2010

Instrumental Analysis of Intrinsically Disordered Proteins

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Challenges for the modern analytical ultracentrifuge analysis of polysaccharides

Cited by 41 publications

References 88 publications

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

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

Accounting for Solvent Signal Offsets in the Analysis of Interferometric Sedimentation Velocity Data

Analytical Ultracentrifugation, a Useful Tool to Probe Intrinsically Disordered Proteins

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