Comparative Snake Venom Analysis with RP-HPLC

A recent article in Royal Society Open Science (1) presented the first comparative analysis of venoms from melanistic (an increased amount of black or nearly black pigmentation [2]) and normally colored common adders (Vipera berus). Researchers compared the venom profiles by sodium dodecylsulfate polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography (RP-HPLC) and tested the venoms’ protease, phospholipase A2, and cytotoxic activities.

The researchers stated in their article that the common adder is the most widespread venomous snake in Europe. The species, and particularly its venom, are mentioned in European folklore, especially across German-speaking countries, with melanistic adders believed to be more aggressive, and more toxic, than adders exhibiting lighter color phenotypes. In fact, these darker adders were once considered to be a separate species called the “hell adder” (3,4), which largely reflects superstitions connected to dark animals, including cats, dogs and ravens, which have been integrated into myths and folklore, often as symbols of bad luck, treachery, evil, and witchcraft (5). While it is now understood that “hell adders” are simply melanistic animals, in certain parts of Europe, such as the more rural parts of Germany, these adders, despite lacking scientific basis, are still feared as more toxic (1). 

Venom was pooled from nine adult males of each phenotype (melanistic or normally colored) and freeze-dried. Only male individuals of German origin were used for this study.

Compositional venom profiling was carried out by the research by combining reducing and non-reducing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 5 µg of each sample and reversed-phase high performance liquid chromatography (RP-HPLC) with 125 µg of each sample. comparison of each phenotype’s venom and melanistic phenotypes (MEL) by SDS-PAGE and RP-HPLC venom profiles revealed qualitative similarities (for example, the number of bands and peaks did not differ between the two venoms), but quantitative differences (for example, bands and peaks presented different intensities between the two venoms) (1).

The researchers concluded that the venoms from the melanistic specimens appeared to display higher protease activity and higher cytotoxicity, though only at a narrow concentration range. While, at first glance, these results support a conceptual difference between venoms of both phenotypes, they only correspond to a few factors tested, and, especially for the cytotoxicity assays, are only detected at lower concentrations they are unlikely to be of clinical relevance. Considering the tremendous extent of venom variation reported in V. berus across its distribution range, the differences observed by the research team may merely be a representation of the normal biological variability within the species rather than a trait of the melanistic animals. The authors of this study recommend further investigations of that topic, using larger sample sizes and additional assays to fully resolve this question. However, the interpretation of the data in light of this known venom variability, the limited range of experiments returning significant differences, and the magnitude of differences measured, it appears that the reputation of the melanistic phenotypes is not based on human experience of envenomation and is probably an irrational superstition after all (1).

Common European adder or common European viper (Vipera berus) basking. © Henri - stock.adobe.com

References

1. Schulte, L.; Uhrig, L.; Eichberg, J.; Schwartze, M.; Auth, I.; Schulz, M.; Lindner, T.; Hien, P.; Hardes, K.; Vilcinskas, A.; Lüddecke, T. Comparative Venom Analysis Between Melanistic and Normally Coloured Phenotypes of the Common Adder (Vipera berus). R. Soc. Open Sci. 2024, 11 (9), 241268. DOI: 10.1098/rsos.241268

2. Melanism definition. https://www.merriam-webster.com/dictionary/melanism (accessed 2024-09-13).

3. Otte, N.; Bohle, D.; Thiesmeier, B. Die Kreuzotter: ein Leben in ziemlich festen Bahnen: mit 105 Abbildungen, 29 Tabellen und 48 Farbtafeln. 2020, Laurenti Verlag.

4. Blum I . 1888 Die Kreuzotter und ihre Verbreitung in Deutschland. Frankfurt am Main. DOI: 10.5962/bhl.title.16086

5. Byghan, Y. Sacred and Mythological Animals: A Worldwide Taxonomy; McFarland, 2020.