4711897_monash_155896.pdf (1.41 MB)
A pharmacological characterisation of cobra and black snake venoms
thesisposted on 2017-03-02, 01:26 authored by Kornhauser, Rachelle Sarah
The current study examined the venoms of the following eight species of snakes: Naja haje, Naja kaouthia, Naja melanoleuca, Naja mossambica, Naja nigricollis, Naja siamensis, Naja sputatrix and Pseudechis australis. The first seven species are cobras, from the genus Naja, and are found throughout Africa and Asia, while the king brown/mulga snake (P. australis) is native to Australia. Cobras (Naja spp.) are a medically important species of snake as they are one of the major contributors to the incidence of snakebite in Africa and Asia. In the current study, a pharmacological profile of the neurotoxic and coagulant activity of each of the seven snakes was established. The data indicated that the rank order neurotoxic potency (based on t90 values), from most to least potent, was: N. kaouthia > N. sputatrix > N. melanoleuca > N. haje > N. mossambica > N. siamensis > N. nigricollis. Further studies also confirmed the presence of anticoagulant proteins in all seven Naja spp. venoms tested. Following from these preliminary findings, experiments examining the in vitro effectiveness of antivenoms were completed using Egyptian cobra (Naja haje) venom, as this snake has been documented to come in contact regularly with humans in northern Africa (i.e. it is the snake of choice for charmers and street performers). Antivenom cross-reactivity (also known as paraspecificity or cross-neutralisation) is when an antivenom raised against a distinct snake venom is effective in neutralising the venom from an unrelated species. In these experiments, native commercial antivenom (i.e. CSL Ltd) raised against the tiger snake (Notechis scutatus) effectively neutralised the neurotoxic activity of Egyptian cobra venom. Likewise, antivenom raised against the Egyptian cobra was able to neutralise the neurotoxic effect of tiger snake venom. This may be due to immunological and biochemical similarities within the elapid family of snakes. Subsequently, an α-neurotoxin (α-elapitoxin-Nh1) was isolated from the Egyptian cobra venom and was pharmacologically characterised. α-Elapitoxin-Nh1 abolished nerve-mediated twitches with a t90 value of one third the time of the whole venom (i.e. ~12 min versus ~38 min). Moreover, α-elapitoxin-Nh1 inhibited cumulative concentration-response curves to carbachol in the unstimulated chick biventer cervicis nerve-muscle preparation with a calculated pA2 value of 8.2, i.e. approximately 80 times more potent that d-tubocurarine. The molecular weight of α-elapitoxin-Nh1 was 6812 Da, and partial amino acid sequencing showed a high sequence homology with other elapid toxins isolated from Naja spp. venoms, as well as toxins from Australian elapids such as α-scutoxin-1 (O. s. scutellatus) and oxylepitoxin-1 (O. microlepidotus). The clinical outcomes due to the presence of anticoagulant proteins in snake venoms can be significant after snakebite, yet the mechanism behind these effects is still not fully understood. To gain further insight into these toxins, an anticoagulant protein (Ac-Pa1) was isolated from the venom of the king brown (mulga) snake (Pseudechis australis). Ac-Pa1 had a molecular mass of 13,128 Da with PLA₂ activity of 262 ± 6 U/ml. Ac-Pa1 inhibited the effect of innovin, (lyophilised recombinant human tissue factor) on normal plasma and also prolonged aPTT. CSL black snake antivenom inhibited the anticoagulant effect of Ac-Pa1. The full amino acid sequence of Ac-Pa1 showed it to be highly homologous with previously isolated enzymes from the king brown snake, as well as toxins isolated from Notechis spp. and Naja spp. venoms, albeit with significant amino acid substitutions at key points central to the structure function relationship of this particular class of toxin. It is evident that amino acid substitutions in key locations within a protein sequence of a snake toxin have a marked impact on the biochemical and pharmacological activity of that toxin, and possibly the whole venom. This research is the first to pharmacologically compare seven (N. haje, N. kaouthia, N. melanoleuca, N. mossambica, N. nigricollis, N. siamensis and N. sputatrix) different cobra venoms from both spitting and non-spitting species, is important in the contribution of snakebite envenoming and treatment with appropriate antivenom, and further serves as a pathway for new drug discoveries and targets using snake venoms and toxins.