Hyaluronan forms specific stable tertiary structures in aqueous solution: A C-13 NMR study

C-13 NMR spectra of aqueous solutions of hyaluronan (HA) of high molecular mass, before and after digestion with testicular hyaluronidase, and of hyal uronan methyl ester were obtained at 125.8 MHz, Carbonyl peaks were assigne d by using selective decoupling techniques, Spectra of digested and undiges ted HA showed sharp signals, except for that assigned to the acetamido carb onyl carbon in the high polymer, which was much broadened. The decreased mo bility of this C=O, thus demonstrated, was caused by restricted rotation. A s part of the rigid CO-NH unit, rotation of NH was therefore similarly rest ricted, probably because of an intermolecular H bond from NH to carboxylate groups on neighbouring HA molecules. This bond was confirmed by comparing esterified HA with unmodified HA. Methyl esterification of carboxylates was accompanied by changes in acetamido C=O resonances consistent with increas ed mobility of CO-NH groups. Ester C=O resonances were sharp, proving that they did not participate in sterically restricted structures such as the pr oposed H bonds involving unesterified carboxylate groups. C=O resonances re port on the environments and on the interrelationships of amide and carboxy late groups. A detailed structure suggested for high-molecular-mass HA in a queous solution takes account of NMR and x-ray fiber diffraction data, Anti parallel HA chains overlap in meshworks stabilized by specific H bonds and hydrophobic bonds. This highly cooperative structure, formally equivalent t o beta-sheets seen in proteins, is not stable in low-molecular-mass HA solu tion. The results relate to structures proposed for shape modules in extrac ellular matrix involving chondroitin and keratan sulfates, which resemble H A in their stereochemistry.