Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to examine the influence of electrolytes (NaCl, KCl, MgCl2, MnCl2 and CaCl2) on the network and hydrodynamic properties of fluoresceinamine-labelled hyaluronan (FA-HA) at concentrations up to 10mg/ml. Self and tracer lateral diffusion coefficients showed that in Ca2+ and Mn2+, FA-HA (830kDa) was more compact than in Mg2+, Na+ or K+. These results were correlated with changes in the hydrodynamic radius of HA, determined by multi-angle laser-light-scattering analysis in dilute solution, which was smaller in CaCl2 (36nm) than in NaCl (43nm). The permeability of more concentrated solutions of HA (< 10mg/ml) to FITC-dextran tracers (2000kDa) was higher in CaCl2. The properties of HA in urea (up to 6M) were investigated to test for hydrophobic interactions and also in ethanol/water (up to 62%, v/v). In both, there was reduced hydrodynamic size and increased permeability to FITC-dextran, suggesting increased chain flexibility, but it did not show the changes predicted if chain–chain association was disrupted by urea, or enhanced by ethanol. Oligosaccharides of HA (HA20–26) also had no effect on the self diffusion of high-molecular-mass FA-HA (830kDa) solutions, or on dextran tracer diffusion, showing that there were no chain–chain interactions open to competition by short-chain segments. The results suggest that the effects of electrolytes and solvent are determined primarily by their effect on HA chain flexibility, with no evidence for association between chain segments contributing significantly to the major properties.

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