Isolation and characterisation of acanmyotoxin-2 and acanmyotoxin-3, myotoxins from the venom of the death adder Acanthophis sp. Seram

Death adder (genus Acanthophis) venoms display neurotoxic activity but were thought to be devoid of myotoxic components. Studies from our laboratory have shown that some species (i.e. Acanthophis rugosus and Acanthophis sp. Seram) posses venom with myotoxic activity [Wickramaratna JC, Fry BG, Aguilar M, Kini RM, Hodgson WC. Isolation and pharmacological characterisation of a phospholipase A2 myotoxin from the venom of the Irian Jayan death adder (A. rugosus). Br J Pharmacol 2003;138:333-342; Wickramaratna JC, Fry BG, Hodgson WC. Species-dependent variations in the in vitro myotoxicity of death adder (Acanthophis) venoms. Toxicol Sci 2003;74:352-360]. The present study describes the isolation and characterisation of two myotoxins (acanmyotoxin-2 and acanmyotoxin-3) from A. sp. Seram venom. Venom was fractionated into approximately 12 major peaks using reverse phase high performance liquid chromatography. Two components caused concentration (0.1-1 μM) dependent inhibition of direct (2 ms, 0.1 Hz, supramaximal V) twitches and an increase in baseline tension in the chick biventer cervicis nerve-muscle. Histological examination of the muscle confirmed damage. PLA2 activity was detected in both acanmyotoxin-2 (390.2 ± 19.7 μmol/(min mg); n = 4) and acanmyotoxin-3 (14.2 ± 7.7 μmol/(min mg); n = 4). In comparison, A. sp. Seram whole venom had a specific activity of 461.3 ± 90.4 μmol/(min mg) (n = 3). Mass spectrometry analysis indicated acanmyotoxin-2 had a mass of 13,082 Da and acanmyotoxin-2 13,896 Da. Acanmyotoxin-2 and acanmyotoxin-3 accounted for approximately 7 and 4% of total venom composition, respectively. N-terminal sequencing of the first 30 amino acids of each toxin indicated they shared some sequence homology with known myotoxins. In conclusion, clinicians should be aware that symptoms of envenoming by some species of death adder may include signs of myotoxicity as well as neurotoxicity. Future studies will investigate the efficacy of the current antivenom treatment against the myotoxic components of A. sp. Seram venom.