Abstract
Acanthophis antarcticus, commonly known as the death adder, is a venomous Australian snake and a member of the Elapidae family. Due to its robust body and triangular head, it was historically misclassified as a viper. Its venom is known for neurotoxic, hemorrhagic, and hemolytic effects but displays low anticoagulant activity. Although key toxins such as three-finger toxins (3FTxs) and phospholipase A(2) (PLA(2)) have been previously described, no study has integrated proteomic and functional analyses to date. In this study, we conducted a comprehensive characterization of A. antarcticus venom. Reverse-phase high-performance liquid chromatography (RP-HPLC) followed by LC-MS/MS enabled the identification of nine toxin families, with 3FTxs and PLA(2) as the most abundant. Less abundant but functionally relevant toxins included Kunitz-type inhibitors, CRISP, SVMP, LAAO, NGF, natriuretic peptides, and nucleotidases, the latter being reported here for the first time based on proteomic evidence. Hydrophilic interaction chromatography (HILIC) coupled with MALDI-TOF was used to analyze polar, non-retained venom components, revealing the presence of low-molecular-weight peptides (2-4 kDa). Functional assays confirmed the enzymatic activity of HYAL, PLA(2), and LAAO and, for the first time, demonstrated inhibitory activity on serine peptidases and fibrinogenolytic activity in the venom of this species. These findings expand our understanding of the biochemical and functional diversity of this venom.