A substrate-gel assay for hyaluronidase activity.

Hyaluronic acid (HA) is a key structural element of the extracellular matrix. Turnover rates of HA are determined in part by hyaluronidases, that are themselves modulated by hyaluronidase inhibitors. A substrate polyacrylamide gel electrophoresis procedure is described here that separates enzyme from inhibitors. The HA is embedded in the gel, and following electrophoretic separation, enzymatic digestion of the HA is allowed to occur. The gel is stained with Alcian blue and can be secondarily stained with Coomassie blue. Enzymatic activities appear as cleared bands on a light blue background, while major proteins appear as dark blue bands. The procedure can be performed in the presence or absence of sodium dodecylsulfate, though levels of hyaluronidase activity decrease when the detergent is used. Hyaluronidases active in the neutral or acid pH range can be detected. This technique will facilitate characterization of hyaluronidases and inhibitors from a wide variety of sources.