Analytical ultracentrifugation is a versatile approach for analysing the molecular mass, molecular integrity (degradation/aggregation), oligomeric state and association/dissociation constants for ...self-association, and assay of ligand binding of kinase related membrane proteins and glycans. It has the great property of being matrix free—providing separation and analysis of macromolecular species without the need of a separation matrix or membrane or immobilisation onto a surface. This short review—designed for the non-hydrodynamic expert—examines the potential of modern sedimentation velocity and sedimentation equilibrium and the challenges posed for these molecules particularly those which have significant cytoplasmic or extracellular domains in addition to the transmembrane region. These different regions can generate different optimal requirements in terms of choice of the appropriate solvent (aqueous/detergent). We compare how analytical ultracentrifugation has contributed to our understanding of two kinase related cellular or bacterial protein/glycan systems (i) the membrane erythrocyte band 3 protein system—studied in aqueous and detergent based solvent systems—and (ii) what it has contributed so far to our understanding of the enterococcal VanS, the glycan ligand vancomycin and interactions of vancomycin with mucins from the gastrointestinal tract.
The analytical ultracentrifuge (AUC) invented by T. Svedberg has now become an extremely versatile and diverse tool in Biochemistry and Polymer Science for the characterisation of the sizes, shapes ...and interactions of particles ranging in size from a few nanometres to tens of microns, or in molecular weight,
M
(molar mass) terms from a few hundred daltons to hundreds of megadaltons. We illustrate this diversity by reviewing recent work on (1) small lignin-like isoeugenols of
M
~ 0.4–0.9 kDa for archaeological wood conservation, (2) protein-like association of a functional amino-cellulose
M
= 3.25 kDa, (3) a small glycopeptide antibiotic (
M
~ 1.5 kDa) and its association with a protein involved in antibiotic resistance (
M
~ 47 kDa), (4) tetanus toxoid protein TTP (
M
~ 150 kDa) and (5) the incorporation of TTP into two huge glycoconjugates considered in glycovaccine development with molecular weight species in a broad distribution appearing to reach 100 MDa. In illustrating the diversity, we will highlight developments in hydrodynamic analysis which have made the AUC such an exciting and important instrument, and point to a potential future development for extending its capability to highly concentrated systems.
We recall the experimental approaches involved in the discovery of hydrogen bonds in deoxyribonucleic acid (DNA) made 70 years ago by a team of scientists at University College Nottingham led by J.M. ...Gulland, and in relation to previous studies. This discovery proved an important step in the elucidation of the correct structure for DNA made by J.D. Watson and F.H.C. Crick, as acknowledged in '
'. At that time of the discovery, however, it was impossible to delineate between inter- and intra-chain hydrogen bonds. We also consider in the light of more recent hydrodynamic theory a tentative model for DNA proposed by Gulland's and D.O. Jordan's PhD student J.M. Creeth in his PhD thesis of 1948, with the correct prediction of two chains with a sugar-phosphate backbone on the exterior and hydrogen-bonded bases between the nucleotide bases of opposite chains in the interior. Our analysis shows that his incorporation of alternating breaks in the two-chain structure was not necessary to explain the viscosity data on scission of hydrogen bonds after titrating to high or low pH. Although Creeth's model is a depiction of DNA structure alone, he could not know whether the hydrogen bonding was intermolecular, although this was subsequently proved correct by others. The mechanisms by which replicative processes occurred were of course unknown at that time, and so, he could not have realised how closely his tentative model resembled steps in some viral replicative mechanisms involving the molecule of life that he was working on.
This work investigated a sustainable and efficient approach of pectin extraction for banana peel waste valorisation and studied the influence of banana ripening stages (RS at 2,5 and 7). Although ...pectin content in banana peel raw material decreased during ripening, pectin extraction was favoured. The highest alcohol-insoluble solids (AIS) yield (12.5%) was achieved at 70 °C, 15 mins from RS 7 peel. All extracts were homogalacturan-rich with some rhamnogalacturonan-I content (showing HGA/RG-I ratio > 2) with varied degree of methylation (DM). The highest HGA content (837.2 mg/g AIS) and HGA/RG-I ratio (9.9) were achieved at 110 °C, 0 mins from RS 7, suggesting its promising application as gelling agent. The highest RG-I content (111.1 mg/g AIS) were obtained at 110 °C, 5 mins from RS 7, which was comparable with the pectin with reported prebiotic ability isolated from the literature, suggesting its potential application in novel products.
•Dramatic decrease of pectin content in raw banana peel during ripening.•Pectin extraction favoured from banana peel at higher ripening stage.•Mild extraction condition thus potential energy saving using ripened banana peel.•Hydrothermal water extraction preserved both pectin HGA and RG-I regions.•Scalable process enabling traditional and novel pectin application.
Stephen Harding, Director of the National Centre for Macromolecular Hydrodynamics based in the UK, is invited to introduce himself as a new Executive Editor of
Biophysical Reviews.
Controlling the rate of softening to extend shelf life was a key target for researchers engineering genetically modified (GM) tomatoes in the 1990s, but only modest improvements were achieved. ...Hybrids grown nowadays contain 'non-ripening mutations' that slow ripening and improve shelf life, but adversely affect flavor and color. We report substantial, targeted control of tomato softening, without affecting other aspects of ripening, by silencing a gene encoding a pectate lyase.
We report the novel application of Analytical Ultracentrifugation (AUCF) to characterise the polymeric proanthocyanidin fraction of hops. Extraction of hop samples with 70% acetone (aq) followed by a ...C-18 Solid Phase Extraction yielded polyphenolic fractions for AUCF analysis. Sedimentation velocity experiments demonstrated the presence of discrete molecular weight bands of proanthocyanidins, as opposed to a continuous distribution of molecular weights. There were 4 such bands for Saaz hop (0.15, 1.1, 2.7 and 4.4S) and 3 bands for Magnum (0.15, 1.6 and 3.0S). The method resulted in a reproducible size (sedimentation coefficient) distribution for replicate runs of the same extract and for extracts prepared from different samples of the same hop variety. Sedimentation equilibrium experiments were then used to fit molecular weight distributions using the new SEDFIT-MSTAR method for the same samples. Thus we report for the first time polymeric proanthocyanidins in hops with molecular weights of up to 100 kDa in Saaz hop (or up to 56 kDa in Magnum). This represents the first application of AUCF to characterise complex fractions of polyphenolics extracted from botanical sources and the methodology developed should find wider application in the study of this diverse and bioactive class of compounds.
Waterlogged archaeological wood is often in need of consolidation prior to drying to prevent shrinkage and cracking of the object. There is a need for new greener materials (than for example ...polyethylene glycol) and methods for consolidation to be developed. The use of wood-based components could provide good interaction between the consolidant and the remaining wood structure and would also support a shift away from fossil fuel-based materials to those with more sustainable sources. Based on this, lignin-like structures have been investigated for their ability to consolidate waterlogged archaeological wood. The in situ formation of a lignin-like material has been carried out using isoeugenol polymerised by horse radish peroxidase in aqueous solution. The formation of the oligomeric/polymeric materials within the wood following this reaction has been determined by Attenuated Total Reflectance Fourier Transform Infra Red (ATR-FTIR) spectroscopy. The oligomers remaining in solution have been characterised by ATR-FTIR and nuclear magnetic resonance (NMR) spectroscopy as well as analytical ultracentrifugation, showing that they have a weight average M
of 0.4-0.9 kDa and a lignin-like structure rich in the β-5' moiety. Therefore, this approach is proposed as a basis to further develop a green consolidation method for waterlogged archaeological wood.
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•Water offers superior performance of okra pectin extraction than acid and alkali.•Homogalacturonan contributes strongly to the flocculating ability of okra extract.•Polymer bridging ...mechanism underpins the flocculant action of okra extract.•A linear relationship between flocculating ability and pectin structure was found.
Okra extract is known to have potential application as a bio-flocculant for wastewater treatment. However, no research to date has given insight into the components responsible for the flocculating ability of okra extract or its flocculating mechanism. The work presented here addresses this knowledge gap showing that pectin, especially pectin homogalacturonan (HGA) regions, appear to be the polysaccharides responsible for the flocculating ability of okra extract. The way pectin works in flocculation may be best explained by a polymer bridging mechanism. Specifically, a linear relationship between okra bio-flocculating ability and pectin homogalacturonan region to rhamnogalacturonan-I region weight ratio (HGA/RG-I) was found (y = 2.0x+47.6, R2 = 0.93, when GalA content > 300 mg/g extract), which was also validated using commercial citrus peel pectin.
Glycopeptide antibiotics are regularly used in ophthalmology to treat infections of Gram-positive bacteria. Aggregative interactions of antibiotics with mucins however can lead to long exposure and ...increases the risk of resistant species. This study focuses on the evaluation of potential interactions of the last line of defence glycopeptide antibiotic teicoplanin with an ocular mucin model using precision matrix free hydrodynamic and microscopic techniques: sedimentation velocity in the analytical ultracentrifuge (SV-AUC), dynamic light scattering (DLS) and atomic force microscopy (AFM). For the mixtures of teicoplanin at higher doses (1.25 mg/mL and 12.5 mg/mL), it was shown to interact and aggregate with bovine submaxillary mucin (BSM) in the distributions of both sedimentation coefficients by SV-AUC and hydrodynamic radii by DLS. The presence of aggregates was confirmed by AFM for higher concentrations. We suggest that teicoplanin eye drop formulations should be delivered at concentrations of < 1.25 mg/mL to avoid potentially harmful aggregations.
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