Reverse hyaluronan substrate gel zymography procedure for the detection ofhyaluronidase inhibitors

Little is known of the ubiquitous inhibitors of hyaluronidase, molecules th at may be important for the deposition of hyaluronan. A reverse hyaluronan- substrate gel procedure is described here that detects such inhibitors, eve n in crude biological extracts, and is independent of the catalytic mechani sm of the target enzyme. Following electrophoresis, hyaluronan-containing g els are incubated in a hyaluronidase solution. Alcian blue-staining bands i ndicate hyaluronan protected from degradation and the location of hyaluroni dase inhibitors. Coordinated use of hyaluronan substrate gel and reverse su bstrate gel procedures provides estimates of the number and relative molecu lar sizes of both enzymes and their inhibitors.