FRACTIONATION OF NORTHERN COPPERHEAD VENOM BY ION-EXCHANGE CHROMATOGRAPHY - PRELIMINARY CHARACTERIZATION OF THE PRIMARY LETHAL FRACTION

J. B. Moran, S. Y. Y. Pang, D. W. Martin and C. R. Geren. Fractionation of northern copperhead venom by ion-exchange chromatography: preliminary characterization of the primary lethal fraction. Toxicon 17, 499-510, 1979.-Venom of the northern copperhead (Agkistrodon contortrix mokasen) has been fractionated by ion-exchange chromatography and the resulting fractions examined for lethal and enzymatic activities. Of the nine distinct fractions obtained by carboxymethyl cellulose chromatography, five were lethal to mice. One of these five accounted for 65-70% of the lethality of whole venom. This fraction, TII, under our experimental conditions appeared to be dissociated from enzymatic activities found both in the whole venom and in other carboxymethyl fractions. Phospholipase, protease and ester-hydrolytic activities appeared in significant quantities in both the fraction of the venom not retained by carboxymethyl cellulose and in various fractions which were retained. Re-chromatography of the non-retained fraction showed that these activities were distinct and not the result of a column overload. The non-retained fraction contained virtually all of the l-amino acid oxidase activity. This fraction also contained a component which caused hemorrhage in mice and a calcium-independent thrombin-like pro-coagulant activity. This fraction was further separated by diethylaminoethyl cellulose chromatography which revealed that the pro-coagulant activity consisted of at least two separate entities. TII had an ld50 value in mice of 1-2 μg/g body weight, which is 10-20 times more potent than whole venom. This fraction has been shown to abolish the muscle response to indirect stimulation and acetylcholine, but not KCl in the chick biventer cervicis neuromuscular junction assay. Its lethality to mice is destroyed by incubation with proteases or at high pH. It is fairly heat stable with lethality being retained after 20 min at 100°C. Toxicity is lost when this fraction is dialyzed against 5 mM sodium acetate, but not when dialyzed against Tyrode's solution or 100 mM sodium acetate. Chemical modification studies have shown that the lethal activity of this fraction may have disulfide, tryptophan, histidine and possibly arginine dependency. © 1979.