A complete set of hyaluronan fragments obtained from hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluronan mass distribution by simple HPLC devices
Abstract
Hyaluronan (HA) has different biological functions according to its molar mass; short HA fragments are involved in inflammation processes and angiogenesis, whereas native HA is not. Physicochemically, studies of native HA hydrolysis catalyzed by bovine testicular hyaluronidase (HAase) have suggested that kinetic parameters depend on HA chain length. To study the influence of HA chain length in more detail, and to try to correlate the physicochemical and biological properties of HA, HA hydrolysis catalyzed by HAase was used in a new procedure to obtain HA fragments of different molar masses. HA fragments (10-mg scale) with a molar mass from 800 to 300,000 g mol−1 were prepared, purified using low-pressure size exclusion chromatography (SEC), lyophilized, and characterized in molar mass by either mass spectrometry or HPLC–SEC–multiangle laser light scattering. The polydispersity index of the purified fractions was less than 1.25. The complete set of HA standards obtained was used to calibrate our routine HPLC–SEC device using only a refractive index (RI) detector. We showed that the N-acetyl-d-glucosamine reducing end assay and the calibrated HPLC–SEC–RI gave equivalent kinetic data. In addition, the HPLC–SEC–RI furnished the mass distribution of the polysaccharide during its hydrolysis.