A complete hyaluronan hydrodynamic characterization using a size exclusion chromatography-triple detector array system during in vitro enzymatic degradation

Size exclusion chromatography coupled with triple detection (online laser light scattering, refractometry, and viscosimetry) (SEC-TDA) was applied for the study of hyaluronan (HA) fragments produced during hydrolysis catalyzed by bovine testicular hyaluronidase (BTH). The main advantage this approach provides is the complete hydrodynamic characterization without requiring further experiments. HA was hydrolyzed using several BTH amounts and for increasing incubation times. Fragments were characterized in terms of weight and number average molecular weights (M_w and M_n, respectively), polydispersity index (M_w/M_n), hydrodynamic radius (R_h), and intrinsic viscosity (η]). The Mark-Houwink-Sakurada (MHS) curves (log η] versus log M_w) were then derived directly. Fragments covering a whole range of M_w (10-900 kDa) and size (R_h = 4-81 nm) and presenting a rather narrow distribution of molar masses (M_w/M_n = 1.6-1.7) were produced. From the MHS curves, HA conformation resulted in a change from a random coil toward a rigid rod structure while decreasing the M_w. HA enzymatic hydrolysis in the presence of a BTH inhibitor was also monitored, revealing that inhibition profiles are affected by ionic strength. Finally, a comparison of the kinetic data derived from SEC-TDA with the data from rheological measurements suggested different strengths of the two methods in the determination of the depolymerization rate depending on the hydrolysis conditions.