Comp. Biochem. Physiol. Vol. 95B, No. 3, pp. 577-582, 1990 0305-0491/90$3.00+ 0.00 Printed in Great Britain © 1990PergamonPress plc A COMPARATIVE STUDY OF THE BIOLOGICAL ACTIVITIES OF VENOMS FROM SNAKES OF THE GENUS AGKISTRODON (MOCCASINS AND COPPERHEADS) NGET-HONG TAN and GNANAJOTHYPONNUDURA! Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia (Received 12 June 1989) Abstract--l. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phospho- monoesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 31 samples of venom from three species of Agkistrodon (A. bilineatus, A. contortrix and A. piscivorus) and 10 venom samples from five other related species belonging to the same tribe of Agkistrodontini were examined. 2. The results indicate that interspecific differences in certain biological activities of the Agkistrodon venoms are more marked than individual variations of the activities, and that these differences can be used for differentiation of the species. Particularly useful for this purpose are the phosphodiesterase, arginine ester hydrolase and anticoagulant activities of the venoms. 3. Venoms of the subspecies of ,4. contortrix and A. piscivorus do not differ significantly in their biological activities. INTRODUCTION Snakes of the genus Agkistrodon (moccasins and copperheads) are rattleless pit vipers and are found in northern and central America. This genus contains three species, A. bilineatus, A. contortrix and A. piscivorus, which contain three, five and three subspe- cies, respectively (Hoge and Hoge, 1978). Snakes of this genus are closely related to snakes from the other four genera of the tribe Agkistrodontini (Hoge and Hoge, 1978), i.e., Calloselasma, Deinagkistrodon, Gloydius and Hypnale. Indeed, many species of snakes from these genera were formerly grouped under the genus Agkistrodon. Many authors suggested that venom properties might be used for the differentiation and classification of snake species (Goncalves and Deutsch, 1956; Tu et al., 1965; Jimenez-Porras, 1967), while others suggested that venom properties have limited value as taxonomic criteria due to intraspecific (individual) variation in venom composition (Johnson, 1968; Soto et al., 1988). While intraspecific differences in venom properties are well documented (Iwanaga and Suzuki, 1979), we have demonstrated that for the genera Naja and Trimeresurus, the magnitude of interspecific differences in certain venom activities is greater than that of intraspecific differences and that interspecific differences in biological activities of Trimeresurus and Naja venoms could be used for differentiation of species (Tan and Tan, 1988a; Tan et al., 1989). Moran and Geren (1979a,b) have examined sub- specific variations in A. contortrix venoms and re- ported major subspecific differences in the amount of distribution of enzymatic activities in ion exchange chromatographic fractions of the venoms. In this paper, we compared the enzymatic, procoagulant, anticoagulant and hemorrhagic activities of 31 sam- pies of venoms from all three species (including eight of the 11 subspecies) of Agkistrodon and examined the possibility of using interspecific differences in venom activities to differentiate between the three Agkistrodon species. For comparison, the biological activities of 10 samples of venom from five other related species/subspecies of snakes from the other four genera of Agkistrodontini, i.e. Calloselasma, Deinagkistrodon, Gloydius and Hypnale, were also examined. MATERIALS AND METHODS Cephalite for kaolin-cephalin clotting time determination was obtained from bioMerieux, France. All other reagents and substrates were of analytical grade and were purchased from Sigma Chemical Company (St Louis, USA). Venoms A total of 41 venom samples were used in this investiga- tion. These included six venom samples of A. bilineatus bilineatus (Mexican moccasin), five venom samples each of A. piseivorus piscivorus (eastern cottonmouth moccasin) and A. contortrix contortrix (southern copperhead), four venom samples each of A. contortrix mokeson (northern copper- head) and A. piscivorus conanti, three venom samples each of A. contortrix laticinctus (broad-banded copperhead), A. piscivorus leueostoma (western cottonmouth moccasin), Calloselasma rhodostoma (Malayan pit viper) and Gloydius blomhoffi blomhoffi (mamushi), two venom samples from Deinagkistrodon acutus (snorkel pit viper) and one venom sample each of A. contortrix pietigaster (trans-pecos copper- head), Gloydius halys ussuriensis and Hypnale hypnale (Merrem's humpnosed pit viper). The venom samples were obtained from Miami Serpentarium Laboratories (Salt Lake City, USA), Latoxan (Rosans, France), Quality Venoms for Medical Research (Florida, USA), Ophidia Venin (Tavan- nes, Switzerland), Ventoxin (Maryland, USA) and Sigma Chemical Company. Sources of venom are indicated in 577