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. 1999 Oct;77(4):2210–2216. doi: 10.1016/S0006-3495(99)77061-X

The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.

P Gribbon 1, B C Heng 1, T E Hardingham 1
PMCID: PMC1300501  PMID: 10512840

Abstract

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.

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Selected References

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  1. Almond A., Brass A., Sheehan J. K. Deducing polymeric structure from aqueous molecular dynamics simulations of oligosaccharides: predictions from simulations of hyaluronan tetrasaccharides compared with hydrodynamic and X-ray fibre diffraction data. J Mol Biol. 1998 Dec 18;284(5):1425–1437. doi: 10.1006/jmbi.1998.2245. [DOI] [PubMed] [Google Scholar]
  2. Almond A., Brass A., Sheehan J. K. Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data. Glycobiology. 1998 Oct;8(10):973–980. doi: 10.1093/glycob/8.10.973. [DOI] [PubMed] [Google Scholar]
  3. Almond A., Sheehan J. K., Brass A. Molecular dynamics simulations of the two disaccharides of hyaluronan in aqueous solution. Glycobiology. 1997 Jul;7(5):597–604. doi: 10.1093/glycob/7.5.597. [DOI] [PubMed] [Google Scholar]
  4. Burcham T. S., Knauf M. J., Osuga D. T., Feeney R. E., Yeh Y. Antifreeze glycoproteins: influence of polymer length and ice crystal habit on activity. Biopolymers. 1984 Jul;23(7):1379–1395. doi: 10.1002/bip.360230720. [DOI] [PubMed] [Google Scholar]
  5. Cleland R. L. Ionic polysaccharides. II. Comparison of polyelectrolyte behavior of hyaluronate with that of carboxymethyl cellulose. Biopolymers. 1968;6(11):1519–1529. doi: 10.1002/bip.1968.360061102. [DOI] [PubMed] [Google Scholar]
  6. Coleman P. J., Scott D., Abiona A., Ashhurst D. E., Mason R. M., Levick J. R. Effect of depletion of interstitial hyaluronan on hydraulic conductance in rabbit knee synovium. J Physiol. 1998 Jun 15;509(Pt 3):695–710. doi: 10.1111/j.1469-7793.1998.695bm.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Comper W. D., Zamparo O. Hydrodynamic properties of connective-tissue polysaccharides. Biochem J. 1990 Aug 1;269(3):561–564. doi: 10.1042/bj2690561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cowman M. K., Cozart D., Nakanishi K., Balazs E. A. 1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment. Arch Biochem Biophys. 1984 Apr;230(1):203–212. doi: 10.1016/0003-9861(84)90101-2. [DOI] [PubMed] [Google Scholar]
  9. Fujii K., Kawata M., Kobayashi Y., Okamoto A., Nishinari K. Effects of the addition of hyaluronate segments with different chain lengths on the viscoelasticity of hyaluronic acid solutions. Biopolymers. 1996 May;38(5):583–591. doi: 10.1002/(SICI)1097-0282(199605)38:5%3C583::AID-BIP4%3E3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  10. Glabe C. G., Harty P. K., Rosen S. D. Preparation and properties of fluorescent polysaccharides. Anal Biochem. 1983 Apr 15;130(2):287–294. doi: 10.1016/0003-2697(83)90590-0. [DOI] [PubMed] [Google Scholar]
  11. Gribbon P., Hardingham T. E. Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching. Biophys J. 1998 Aug;75(2):1032–1039. doi: 10.1016/S0006-3495(98)77592-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Morris E. R., Rees D. A., Welsh E. J. Conformation and dynamic interactions in hyaluronate solutions. J Mol Biol. 1980 Apr;138(2):383–400. doi: 10.1016/0022-2836(80)90294-6. [DOI] [PubMed] [Google Scholar]
  13. Reed C. E., Li X., Reed W. F. The effects of pH on hyaluronate as observed by light scattering. Biopolymers. 1989 Nov;28(11):1981–2000. doi: 10.1002/bip.360281114. [DOI] [PubMed] [Google Scholar]
  14. Scott J. E., Cummings C., Brass A., Chen Y. Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient network-forming polymer. Biochem J. 1991 Mar 15;274(Pt 3):699–705. doi: 10.1042/bj2740699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Scott J. E., Heatley F., Moorcroft D., Olavesen A. H. Secondary structures of hyaluronate and chondroitin sulphates. A 1H n.m.r. study of NH signals in dimethyl sulphoxide solution. Biochem J. 1981 Dec 1;199(3):829–832. doi: 10.1042/bj1990829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Scott J. E. Supramolecular organization of extracellular matrix glycosaminoglycans, in vitro and in the tissues. FASEB J. 1992 Jun;6(9):2639–2645. [PubMed] [Google Scholar]
  17. Scott J. E., Tigwell M. J. The influence of the intrapolymer environment on periodate oxidation of uronic acids in polyuronides and glycosaminoglycuronans. Biochem Soc Trans. 1975;3(5):662–664. doi: 10.1042/bst0030662. [DOI] [PubMed] [Google Scholar]
  18. Wik K. O., Comper W. D. Hyaluronate diffusion in semidilute solutions. Biopolymers. 1982 Mar;21(3):583–599. doi: 10.1002/bip.360210308. [DOI] [PubMed] [Google Scholar]
  19. Winter W. T., Smith P. J., Arnott S. Hyaluronic acid: structure of a fully extended 3-fold helical sodium salt and comparison with the less extended 4-fold helical forms. J Mol Biol. 1975 Dec 5;99(2):219–235. doi: 10.1016/s0022-2836(75)80142-2. [DOI] [PubMed] [Google Scholar]

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