Secondary structure of hyaluronate in solution. A 1H-n.m.r. investigation at 300 and 500 MHz in [2H6]dimethyl sulphoxide solution

Abstract

The 1H-NMR spectra of solutions in [2H6]dimethyl sulfoxide of the sodium salts of tetra-, hexa- and octa-saccharides prepared from hyaluronate by testicular-hyaluronidase digestion were examined at 300 and 500 MHz. The signals from hydroxy groups at positions 2 and 3 in the glucuronic acid moiety were assigned. Their chemical shifts and associated temperature-dependencies, as well as their coupling constants, depended on whether or not the uronic acid was at the non-reducing end. Deviations from the normal pattern of hydroxy-group proton NMR behavior were attributable to participation in H-bonds, either to the acetamido carbonyl O atom or the pyranose ring O atom of neighboring N-acetylhexosamine moieties. A secondary structure, containing 4 different H-bonds per trisaccharide unit of glucuronosyl-hexosaminyl-glucuronic acid, was demonstrated. This is the first complete and detailed secondary structure to be established for hyaluronate in any solvent. Hyaluronate is compared with chondroitin sulfate, dermatan sulfate, heparan sulfate and keratan sulfate in their potential to form secondary structures with features in common. The significance of the details of the structure to its overall stability and the probability of their persistence into aqueous environments are discussed. The presence of all or most of the secondary structure in glycosaminoglycuronans is correlated with a space-filling function in the tissue, and with a high carbohydrate content in the parent proteoglycan in the case of the chondroitin sulfates.