Evaluation of the Information Content of Sedimentation Equilibrium Data in Self‐Interacting Systems

Ang, Shirley; Rowe, Arthur J.

doi:10.1002/mabi.201000065

Cited by 20 publications

(25 citation statements)

References 13 publications

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“…Running of the algorithm takes only a few minutes, in contrast to the time required previously to analyse sedimentation equilibrium data for polymers, and obviating the difficult and inconvenient procedures for obtaining adequate baselines encountered in earlier studies 2,12 . A new method (MultiSig) for obtaining accurate values for the baseline offset (E) in fringe optics via multi-exponential fitting has recently been published 26 . MultiSig returns the mean of 20 estimates using a Monte-Carlo type approach.…”

Section: Discussion and Perspectivesmentioning

confidence: 99%

“…The removal of TI noise can also be effected experimentally, for interference data, by taking a series of scans immediately at the start of the run, averaging same, and subtracting this averaged set of radial values from the final (usually averaged) data set at equilibrium 26 . This routine has been followed for all the experimental data reported here.…”

Section: Theorymentioning

confidence: 99%

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SEDFIT–MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge

Schuck

Gillis

Besong

et al. 2014

Analyst

121

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Sedimentation equilibrium (analytical ultracentrifugation) is one of the most inherently suitable methods for the determination of average molecular weights and molecular weight distributions of polymers, because of its absolute basis (no conformation assumptions) and inherent fractionation ability (without the need for columns or membranes and associated assumptions over inertness). With modern instrumentation it is also possible to run up to 21 samples simultaneously in a single run. Its application has been severely hampered because of difficulties in terms of baseline determination (incorporating estimation of the concentration at the air/solution meniscus) and complexity of the analysis procedures. We describe a new method for baseline determination based on a smart-smoothing principle and built into the highly popular platform SEDFIT for the analysis of the sedimentation behavior of natural and synthetic polymer materials. The SEDFIT-MSTAR procedure – which takes only a few minutes to perform - is tested with four synthetic data sets (including a significantly non-ideal system) a naturally occurring protein (human IgG1) and two naturally occurring carbohydrate polymers (pullulan and λ–carrageenan) in terms of (i) weight average molecular weight for the whole distribution of species in the sample (ii) the variation in “point” average molecular weight with local concentration in the ultracentrifuge cell and (iii) molecular weight distribution.

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Section: Discussion and Perspectivesmentioning

confidence: 99%

Section: Theorymentioning

confidence: 99%

SEDFIT–MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge

Schuck

Gillis

Besong

et al. 2014

Analyst

121

View full text Add to dashboard Cite

show abstract

“…M w from SEDFIT-MSTAR could then be combined with the weight average sedimentation coefficient to yield the molecular weight distribution54. We also estimate M z using the MFIT algorithm of Ang and Rowe55.…”

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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“…Where the third virial term (3CM) is significant, this can be defined in addition to the second virial term, as defined above. Even a protein so highly soluble as RNase A has been shown to self-associate in the millimolar region and to exhibit a significant third virial term under conditions of low ionic strength [16]. By analogy with the case for strong interactions presented above for the ETF system, the possible presence of irreversibly associated species still needs to be established, by either (i) several runs at different cell-loading concentration c o , and/or (ii) a SV run to define the presence, if any, of such species.…”

Section: Choice Of Optical Systemmentioning

confidence: 99%

“…It is important that the precision of estimates of parameters secured should be defined by a full 'bootstrapping" procedure. The levels of cell loading used are critical for success, and a full definition of these levels as given by computer simulation, and of the precision which can be expected in retrieved parameters has been provided (see [16] and references cited therein).…”

Section: Choice Of Optical Systemmentioning

confidence: 99%

Insight into protein–protein interactions from analytical ultracentrifugation

Harding

Rowe

2010

Biochemical Society Transactions

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Analytical ultracentrifugation is a free solution technique with no supplementary immobilization, columns or membranes required, and can be used to study self-association and hetero-interactions, stoichiometry, reversibility and interaction strength across a very large dynamic range (dissociation constants from 10(-12) M to 10(-1) M). In the present paper, we review some of the advances that have been made in the two different types of sedimentation experiment--sedimentation equilibrium and sedimentation velocity--for the analysis of protein-protein interactions and indicate how major complications such as thermodynamic and hydrodynamic non-ideality can be dealt with.

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Evaluation of the Information Content of Sedimentation Equilibrium Data in Self‐Interacting Systems

Cited by 20 publications

References 13 publications

SEDFIT–MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge

SEDFIT–MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge

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

Insight into protein–protein interactions from analytical ultracentrifugation

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