Quantifying venom in African snakes: Insights into protein content, yield and body size associations

Snake venoms are complex mixtures primarily composed of toxic proteins used during prey capture and defence. There is limited knowledge concerning the protein concentration of snake venom and the biases of different protein determination methods. Here, we assess the ability of the Qubit protein assay, bicinchoninic acid (BCA) assay, Bradford assay and NanoDrop spectrometry (A280 with a mass extinction coefficient of one) to accurately quantify protein concentrations of toxins isolated from venom, including three-finger toxins and phospholipase A₂. The Bradford assays severely underestimated three-finger toxin concentrations and NanoDrop spectrometry overestimated phospholipase A₂ concentrations, whilst the BCA assay was the most accurate. Venom from six major African venomous snake genera was also assessed: coral cobras (Aspidelaps spp.); mambas (Dendroaspis spp.); cobras (Naja spp.); bush vipers (Atheris sp.); adders (Bitis spp.); and saw-scaled vipers (Echis sp.). Protein concentration results were inconsistent between methods. Protein concentrations were found to be lowest for Bitis spp. venom and highest for Naja spp. venom and did not vary between species of the same genus. However, in general, Elapidae species had venoms with significantly higher protein concentrations than Viperidae species. Moreover, there was greater variability between Elapidae species. We also determined wet venom yields and used this to provide a tentative estimate of the total protein quantity that may be injected during a snake bite. We found snake weight and length influenced wet venom yield for the Atheris squamigera but not for Bitis arietans and Echis romani. Our results aim to improve our understanding of the physical properties of snake venom.