A simple assay for hyaluronidase activity using fluorescence polarization

Hyaluronan is a large glycosaminoglycan and is a major constituent of the extracellular matrix, interacting with cell surface receptors such as CD44. We previously reported that fragmented hyaluronan, with the size frequently detected in cancer patients, induces CD44 cleavage and concomitantly enhances tumor cell migration. Although hyaluronan degradation to smaller fragments has been revealed to be a key reaction in regulating cancer progression, simple methods for continuously detecting hyaluronidase activity have not been established. Here, we show that fluorescently-labeled hyaluronan serves as a substrate for continuous assay of hyaluronidase activity. A very simple assay was established to measure degradation of hyaluronan based on fluorescence polarization. The developed assay method would provide a way for continuous measurement of cellular hyaluronidase activity and also for measurement of binding of hyaluronan to its receptors, and thus should be useful for investigation of the function of hyaluronan in cancer progression.