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

13C NMR spectra of aqueous solutions of hyaluronan (HA) of high molecular mass, before and after digestion with testicular hyaluronidase, and of hyaluronan methyl ester were obtained at 125.8 MHz. Carbonyl peaks were assigned by using selective decoupling techniques. Spectra of digested and undigested HA showed sharp signals, except for that assigned to the acetamido carbonyl carbon in the high polymer, which was much broadened. The decreased mobility of this C==O, thus demonstrated, was caused by restricted rotation. As part of the rigid CO---NH unit, rotation of NH was therefore similarly restricted, 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 increased mobility of CO---NH groups. Ester C==O resonances were sharp, proving that they did not participate in sterically restricted structures such as the proposed H bonds involving unesterified carboxylate groups. C==O resonances report on the environments and on the interrelationships of amide and carboxylate groups. A detailed structure suggested for high-molecular-mass HA in aqueous solution takes account of NMR and x-ray fiber diffraction data. Antiparallel HA chains overlap in meshworks stabilized by specific H bonds and hydrophobic bonds. This highly cooperative structure, formally equivalent to beta-sheets seen in proteins, is not stable in low-molecular-mass HA solution. The results relate to structures proposed for shape modules in extracellular matrix involving chondroitin and keratan sulfates, which resemble HA in their stereochemistry.