Characterization of hyaluronidase isolated from Agkistrodon contortrix contortrix (southern copperhead) venom

Snake venoms are a rich source of enzymes including many hydrolytic enzymes . Some enzymes such as phospholipase A(2), proteolytic enzymes, and phospho diesterases are well characterized. However many enzymes, such as the glyco sidase, hyaluronidase, have not been studied extensively. Here we describe the characterization of snake venom hyaluronidase. In order to determine wh ich venom was the best source for isolation of the enzyme, the hyaluronidas e activity of 19 venoms from Elapidae, Viperidae, and Crotalidae snakes was determined. Since Agkistrodon contortrix contortrix venom showed the highe st activity, this venom was used for purification of hyaluronidase. Molecul ar weight was determined by matrix-assisted laser desorption ionization mas s spectroscopy and was found to be 59,290 Da. The molecular weight value as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis wa s 61,000 Da. Substrate specificity studies indicated that the snake venom e nzyme was specific only for hyaluronan and did not hydrolyze similar polysa ccharides of chondroitin, chondroitin sulfate A (chondroitin 4-sulfate), ch ondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D, chondroitin sulfate E, or heparin, The e nzyme is an endo-glycosidase without exo-glycosidase activity, as it did no t hydrolyze p-nitrophenyl-beta -D-glueuronide or p-nitrophenyl-N-acetyl-bet a -D-glucosaminide. The main hydrolysis products from hyaluronan were hexa- and tetrasaccharides with N-acetylglucosamine at the reducing terminal. Th e cleavage point is at the beta1,4-glycosidic linkage and not at the beta1, 3-glycosidic linkage. Thus, snake venom hyaluronidase is an endo-beta -N-ac etylhexosaminidase specific for hyaluronan. (C) 2001 Academic Press.