Identification of PLA2 and α-Neurotoxin Proteins in the Venom of Pseudonaja affinis (Dugite)

Abstract

The Western brown snake Pseudonaja affinis (dugite), common to the Perth area of Western Australia, possesses one of the most lethal venoms in the world. Little is known, however, about the toxic protein constituents of the venom, other than those causing coagulopathic and procoagulant effects. The current study was therefore undertaken in order to identify other protein constituents and activities present. Crude venom induced a contraction in rat tracheal preparations through phospholipase A2 (PLA2) activity, as shown by the complete and partial inhibition of contraction by PLA2 inhibitors 4-bromophenacyl bromide and quinacrine. Further, a reduced degree of smooth muscle contraction in the presence of the leukotriene receptor antagonist SKF104353 suggested that this effect was mediated by leukotriene metabolites. The venom-induced contraction did not reoccur upon a second administration of the venom, despite the muscle retaining its contractile function and appearing histologically undamaged. Chromatographic separation of the protein constituents of the venom showed that PLA2 activity was associated with all protein fractions. A low-molecular-weight component of the venom was further investigated through N-terminal sequencing and found to possess high identity to the short-chain α-neurotoxin family of toxins. Venom activity on cultured rat cardiac myocytes and cultured cortical neurons was also examined. The crude venom was found to temporarily inhibit the beating of the cardiac myocytes, after which the beating resumed erratically. Cortical neurons, however, were irreversibly affected, showing concentration-dependent cell death.