Quantitative bead assay for hyaluronidase and heparinase I

A novel, simple, and sensitive assay was developed to monitor, quantitatively, the hyaluronidase and heparinase I-catalyzed cleavage of fluoresceinamine-labeled hyaluronic acid and heparin, respectively. The fluoresceinamine-labeled substrates were hydrophobically absorbed onto 4-microm polystyrene beads. In the presence of enzyme, the change in fluorescence output of the substrate-absorbed beads was monitored in a noncontinuous manner using a flow cytometer. Our results show that hyaluronidase and heparinase I can cleave their respective substrates on the beads in a concentration- and time-dependent manner. The assay is suitable for detecting the presence of these glycosaminoglycan-degrading enzymes in cell lysates, extracts, or purified fractions, for quantifying their amounts, and for investigating the activity of potential inhibitors.