The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.
Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides.
Gribbon
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Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
Heng
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BC
Hardingham
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D000818:Animals; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D004058:Diffusion / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004573:Electrolytes; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D008956:Models, Chemical; D008970:Molecular Weight; D009994:Osmolar Concentration; D000071228:Polyelectrolytes; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D012972:Sodium Hydroxide / Q000494:pharmacology; D012996:Solutions; D055672:Static Electricity
D003911:Dextrans; D004573:Electrolytes; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006820:Hyaluronic Acid; D000071228:Polyelectrolytes; D011108:Polymers; D012965:Sodium Chloride; D012972:Sodium Hydroxide; D012996:Solutions; C015219:fluorescein isothiocyanate dextran; C030864:5-aminofluorescein