Hyaluronidase inhibition assay

The inhibition activity of hyaluronidase against BUnF, BAW, and BAL was investigated by using a colorimetric assay based on the modified Morgan-Elson method (17–19) which measures the amount of N-acetyl-D-glucosamine produced from sodium hyaluronate. Briefly, 12 µl of BUnF, BAW, or BAL were mixed with 12 µl of hyaluronidase (10 mg/ml) in 0.1 M sodium acetate buffer (pH 3.5), then incubated in a water bath at 37°C. After pre-incubation for 20 min, the sample and hyaluronidase solution mixture was added to 12 µl of 12.5 mM calcium chloride to activate the hyaluronidase and then incubated for 20 min. To the activated hyaluronidase solution, 24 µl of sodium hyaluronate (6 mg/ml) dissolved in 0.1 M sodium acetate buffer (pH 3.5) was added and incubated for an additional 40 min. Next, the solution was amended with 12 µl of 0.4 N NaOH and 0.4 M potassium tetraborate, then vortexed, boiled for 3 min, and placed on ice to terminate the hyaluronidase activity. Subsequently, 360 µl of DMAB solution (p-dimethylaminobenzaldehyde 4 g in a solution of acetic acid 350 ml mixed with 50 ml of 10 N HCl) was added, and the sample placed in a 37°C water bath for 20 min. The absorbance of each test tube at 540 nm was measured by using a microplate reader (Tecan Sunrise, Tecan, Hombrechtikon, Switzerland). Hyaluronidase inhibition activity was estimated by determining differences in the absorbance values of the sample solutions.