Kinetic investigation of recombinant human hyaluronidase PH20 on hyaluronic acid

The kinetic investigation of hyaluronidases using physiologically relevant hyaluronic acid (HA or hyaluronan) substrate will provide useful and important clues to their catalytic behavior and function in vivo. We present here a simple and sensitive method for kinetic measurement of recombinant human hyaluronidase PH20 (rHuPH20) on HA substrates with sizes ranging from 90 to 752 kDa. The method is based on 2-aminobenzamide labeling of hydrolyzed HA products combined with separation by size exclusion–ultra performance liquid chromatography coupled with fluorescence detection. rHuPH20 was found to follow Michaelis–Menten kinetics during the initial reaction time. Optimal reaction rates were observed in the pH range of 4.5–5.5. The HA substrate size did not have significant effects on the initial rate of the reaction. By studying HA substrates of 215, 357, and 752 kDa, the kinetic parameters Km, Vmax, and kcat were determined to be 0.87–0.91 mg/ml, 1.66–1.74 nM s−1, and 40.5–42.4 s−1, respectively. This method allows for direct measurement of kinetics using physiologically relevant HA substrates and can be applied to other hyaluronidase kinetic measurements.