ANALYSIS OF GLYCOSAMINOGLYCAN-DEGRADING ENZYMES BY SUBSTRATE GEL-ELECTROPHORESIS (ZYMOGRAPHY)

Substrate gel electrophoresis is of use for detecting minute amounts o f hyaluronidase (HAase). In substrate gel electrophoresis, hyaluronan (HA) is impregnated in a gel, To determine the presence of degradation enzymes for other glycosaminoglycans (GAGs), the sizes of whose molec ules are much smaller than that of HA, we have developed a technique b y which chondroitin sulfate (CS) is chemically modified by introducing an allyl group at the reducing end for its immobilization in the gel, Enzymes with CS-degrading activity were detected on a CS-copolymerize d gel in the presence or absence of sodium dodecyl sulfate. The smalle st amount of chondroitinase ABC and HAase was found to be 8 mu U and 0 .35 mU, respectively. By zymography using HA-impregnated and modified CS-copolymerized gels human serum HAase has been shown to consist of a t least two isoforms each with its own substrate specificity, Using th is method, uterine tumor tissue has been shown to secrete a novel HAas e which degrades HA at neutral pH, but not CS at any pH, This method w as also confirmed applicable to other GAGs for determining individual GAG-degrading enzymes, In future research, it will be used to examine the regulation of each GAG species in tissue. (C) 1995 Academic Press, Inc.