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 use d to investigate intramolecular hydrogen bonding and electrostatic interact ions in hyaluronan solutions. Self and tracer lateral diffusion coefficient s within hyaluronan solutions were measured over a wide range of concentrat ions (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). R eductions in self- and tracer-diffusion with c followed a stretched exponen tial model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaC l. Disruption of hydrogen bonds by strong alkali (0.6 M NaOH) resulted in f urther larger increases in self- and tracer-diffusion coefficients, consist ent with a more dynamic and permeable network. Concentrated hyaluronan solu tion properties were attributed to hydrodynamic and entanglement interactio ns between domains. There was no evidence of chain-chain associations. At p hysiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides.