Functional and Proteomic Characterization of Acanthophis antarcticus Venom: Evidence of Fibrinogenolytic and Serine Peptidase Inhibitory Activities

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 A₂ (PLA₂) 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 PLA₂ 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, PLA₂, 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.

Keywords: Acanthophis; Elapidae; fibrinogenolytic activity; metallopeptidases; proteomic; serine peptidase inhibitors; serine peptidases.

Figure 1

Reverse-phase (RP) chromatogram of A. antarcticus venom. The figure shows the numbering of the peaks collected for proteomic analysis, as well as the distribution of the toxin classes identified along the chromatographic profile. Legend: 3FTxs (three-finger toxins), PLA₂ (phospholipase A₂), NP (natriuretic peptide), KTI (Kunitz-type inhibitors), NGF (nerve growth factor), CRISP (cysteine-rich secretory protein), SVMP (metalloproteinase), NSase (nucleotidase), and LAAO (L-amino acid oxidase).

Figure 2

(A) Hydrophilic interaction liquid chromatography (HILIC) of A. antarcticus venom samples, previously subjected to solid-phase extraction (SPE). (B) Mass spectrum obtained by MALDI-TOF from fractions collected by HILIC.

Figure 3

Relative quantification and diversity (isoforms) of the protein classes identified in the proteome of A. antarcticus venom. Relative quantification was performed according to the methodology described by [45], and the detailed calculation is provided in Supplementary Material S2 (Table S3). Legend: 3FTxs (three-finger toxins), PLA₂ (phospholipase A₂), NP (natriuretic peptide), KTI (Kunitz-type inhibitors), NGF (nerve growth factor), CRISP (cysteine-rich secretory protein), SVMP (metalloproteinase), NSase (nucleotidase), and LAAO (L-amino acid oxidase).

Figure 4

Panel (A) demonstrates hyaluronidase (HYAL) activity, evidenced by the hydrolysis of hyaluronic acid at different venom concentrations, with isolated hyaluronic acid used as a negative control; experiments were conducted in duplicate, and the results are expressed as mean ± standard deviation. Panels (B,C) show the activities of phospholipase A₂ (PLA₂) and L-amino acid oxidase (LAAO), respectively, with statistically significant differences (p < 0.05), using Bothrops jararaca venom as a positive control. All assays were performed in triplicate, and the results are expressed as mean ± standard deviation. The asterisk (*) indicates statistically significant differences compared to the control group (p < 0.05). Panel (D) shows the fibrinogenolytic activity of the venom, assessed by SDS-PAGE, where the control sample (Fib) displays intact α, β, and γ fibrinogen chains, while the subsequent lanes represent the effects of venom incubation at different concentrations (20 and 40 µg) and reaction times (5, 30, and 90 min). Panel (E) presents the fibrinogenolytic activity of venom incubated with fibrinogen in the presence and absence of the inhibitors PMSF and EDTA, using 40 µg of venom for 90 min, an optimized condition previously established in panel (D). As a negative control, isolated fibrinogen (Fib) is shown with preserved α, β, and γ chains. All samples were analyzed in triplicate by SDS-PAGE. Finally, panels (F,G) illustrate the inhibitory activity against serine peptidase (trypsin) in samples treated with native and heat-denatured venom, respectively, at different concentrations. These assays were performed in duplicate, and the results are expressed as mean ± standard error.