Experimental Design

Venom composition

Snake venom is a complex mixture of proteins, enzymes and other substances with toxic and lethal properties. Its function it to defend against threats, immobilize the prey, and help the digestion. Some of the proteins in the snake venom have very specific effects on biological functions, such as blood coagulation, blood pressure regulation and transmission of nervous or muscular impulses.

Proteins constitute 90-95% of venom’s dry weight and are responsible for most of its biological effects. Enzymes make up 80-90% of the Viperidae family venom and 25-70% of the Elapidae family venom [1]. The most common enzymes are oxidases, phospholipases, metalloproteinases, hydrolases and serine proteases. Polypeptide toxins include cytotoxins (effects mainly on local tissue), cardiotoxins (effects on heart tissue) and neurotoxins (effects on nerve tissue).

Venomic studies have enabled the characterization of venom composition, from snakes species of medical importance [2]. From these, it can be shown that the venom between snake species, even within families, is more similar than expected [3]. Major protein families like metalloproteinases (SVMPs), Phospholipases A2 (PLA2), cysteine rich secretory proteins (CRISPs) are present in all snake families, with the relative abundance in the venom composition being the only difference [4].
However, these studies have also shown that there are certain enzymes that are probably innate to snake families, for example the prominent serine proteases in the Viperidae venom, or the much more abundant three finger toxins (3FTx) in Elapidae venom.

Choice of enzymatic assay

In Australia, immunoassays based on venom-specific antibodies have been developed to distinguish between the most lethal snakes in that region [5]. Immunoassays have been used or suggested to be used as a detection mechanism for snake venom for many years [5,6].

We apply an alternative approach to detect snake venom circumventing immunoassay based methods. We want to create a proteolytic enzyme assay based on a linker sequence which contains a cleavage site for proteases that are characteristic for the venom of different snakes.

Proteins that give off signals when a linker sequence is cleaved, are not a new invention. FRET (Föster resonance energy transfer) has been used for many years in research, to gain a better understanding of protein-protein interactions.

Our approach to snake venom detection, utilising the proteolytic activity of different venoms, has several potential advantages compared to current methods used for snake venom detection:

1. - The recombinant proteins can be produced at a lower price compared to the current immunological methods, which involves inoculating snake venom in horses.
2. - With this approach, we are able to find new potential target peptides for snake venom cleavage in a relatively fast and cheap way. Screening of these peptides is much less time consuming and easier than trying to identify suitable antibodies for the venom enzymes and toxins.
3. - If reliable measurements can be made, we might be able to not only detect which snake people have been bitten by, but also to determine the level of venom injected into the blood.

References

[1] Bauchot, R (1994). Snakes: A Natural History. Sterling Publishing Co., pp. 194–209.
[2] Fasoli E, Sanz L, Wagstaff S, Harrison RA, Righetti PG, Calvete JJ (2010). Exploring the venom proteome of the African puff adder, Bitis arietans, using a combinatorial peptide ligand library approach at different pHs. J Proteomics.,73(5):932-42.
[3]Calderón-Celis F, Cid-Barrio L, Encinar JR, Sanz-Medel A, Calvete JJ (2017). Absolute venomics: Absolute quantification of intact venom proteins through elemental mass spectrometry. J Proteomics., 164:33-42.
[4] Calvete JJ, Marcinkiewicz C, Sanz L (2007). Snake venomics of Bitis gabonica gabonica. Protein family composition, subunit organization of venom toxins, and characterization of dimeric disintegrins bitisgabonin-1 and bitisgabonin-2. J Proteome Res., 6(1):326-36.