Poly-acid properties of biosynthetic hyaluronan studied by titration

In this study the poly-acid properties of biosynthetic hyaluronan produced by fermentation of Bacillus subtilis have been investigated. Potentiometric titration as well as H-1 NMR titration have been used to determine the dissociation constants of the carboxylic group on hyaluronic acid. The intrinsic pK(a) and pK(a, alpha=0.5) were determined in the presence of 0.1 M salt to be 2.99 and 3.37, respectively. The pK(a, alpha=0.5) was found to be unaffected by variations in the ionic strength which is in good agreement with the fact that at alpha = 0.5, 50% of the carboxylic moieties on the hyaluronan are charged. On the other hand the intrinsic pK(a) was found to be dependent on the ionic strength until the Debye-Huckel screening length approaches the length of repeating disaccharide unit of hyaluronic acid. Our findings are in good agreement with previously determined dissociation constants for other sources of hyaluronan. We have also shown that H-1 NMR spectroscopy is the preferred method for polyelectrolyte titration because of the ability to isolate the contribution of several ionisable groups on a polymer on molecular level. (c) 2008 Elsevier Ltd. All rights reserved.