| High performance gel permeation chromatography (HP-GPC) and sensitivity-enhanced polyacrylamide gel electrophoresis (SE-PAGE) have been combined in an analytical procedure for the fractionation and characterization of hyaluronan (HA) and other glycosaminoglycans (GAGs). Preparative-scale application of the method enabled the rapid isolation of low molecular weight HA compounds for aqueous solution-state studies of HA conformation and chain-chain association by {dollar}sp1{dollar}H and {dollar}sp{lcub}13{rcub}{dollar}C NMR spectroscopy.; Analytical application. Enzymatically digested samples of HA, chondroitin 4-sulfate, and dermatan sulfate were fractionated by HP-GPC. Isolated fractions were subjected to SE-PAGE for determination of molecular weight. The column calibration profiles, obtained in 0.15M NaCl, yielded the following data: (1) at equal degrees of polymerization, HA chains have an apparently smaller hydrodynamic radius than the sulfated polymers, and (2) at equal molecular weights, all three GAG's have approximately equal hydrodynamic radii.; 300 MHz {dollar}sp1{dollar}H and 76 MHz {dollar}sp{lcub}13{rcub}{dollar}C NMR. Two of the fractions isolated by the preparative scale application of the above method were utilized as part of the NMR investigations. As determined by SE-PAGE, the weight-average degrees of polymerization of these HA segments samples equaled 40 (HA{dollar}sb{lcub}40{rcub}{dollar}) and 27 (HA{dollar}sb{lcub}27{rcub}{dollar}) disaccharide repeat units, respectively.; A summary of the {dollar}sp{lcub}13{rcub}{dollar}C NMR investigations of samples in aqueous 0.15M NaCl solutions, at or near neutral pH, follows: (1) Chainlength dependent studies indicated that low molecular weight HA segments compounds are an effective model system for high molecular weight polymer HA. (2) Ionic strength dependent changes were seen in the conformation of the {dollar}beta{dollar}-1,3 linkage of HA{dollar}sb{lcub}27{rcub}{dollar} as the NaCl molarity was varied from zero to 0.15M. (3) Chain-chain association between HA{dollar}sb{lcub}27{rcub}{dollar} segments was not observed by one-dimensional {dollar}sp{lcub}13{rcub}{dollar}C or {dollar}sp{lcub}1{rcub}{dollar}H NMR.; {dollar}sp{lcub}1{rcub}{dollar}H NMR was used to investigate the amide proton environment of HA{dollar}sb{lcub}27{rcub}{dollar} and HA{dollar}sb{lcub}40{rcub}{dollar} in aqueous solutions containing no added salt, 0.15M NaCl, or 0.15M KCl. No evidence for a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. |
