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.
Krupa
Joanne C
JC
Joint Diseases Laboratory, Shriners Hospital for Children, Montreal, Que., Canada.
Marie Butler
Ann
A
Mort
John S
JS
D000818:Animals; D002417:Cattle; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D006493:Heparin / Q000031:analogs & derivatives; D019763:Heparin Lyase / Q000032:analysis / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000032:analysis / Q000378:metabolism*; D008297:Male; D008863:Microspheres; D011137:Polystyrenes / Q000737:chemistry; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013737:Testis / Q000201:enzymology
D005452:Fluoresceins; D011137:Polystyrenes; D006820:Hyaluronic Acid; D006493:Heparin; D006821:Hyaluronoglucosaminidase; D019763:Heparin Lyase