ICP-MS characterization of seven North American snake venoms

Highlights

• Used inductively coupled plasma mass spectrometry to count metal ions in snake venom.

• Quantified concentration of 71 metal isotopes from <0.2 to >100,000 PPB

• Observed a negative relationship between magnesium level and subcutaneous toxicity.

Abstract

Snake venoms are inherently complex. They are mixtures of multiple enzymes, peptides, lipids, carbohydrates, nucleosides, and metal ions. Metal ions make up a small portion of a snake's venom but play outsized roles in enzyme function and stability. Unlike enzyme primary structure, which is easily predicted from genomic sequences, a venom's metal ion content must be measured directly. We leveraged the high throughput and sensitivity of inductively coupled plasma mass spectrometry to analyze the metal ion content of seven North American snake venoms. All venoms were collected from snakes reared at one location, so we could discount variation from environmental or geographical factors. We profiled 71 metal isotopes. Selenium isotopes were consistently high across all venoms tested. When each venom's toxicity was graphed as a function of each different metal isotope, the only strong relationships between metal content and toxicity were for magnesium isotopes.

Introduction

Snake venoms are complex mixtures of many parts working synergistically to incapacitate predators or prey through multiple mechanisms such as causing paralysis, intense pain, inhibiting the fight-or-flight response, or disrupting the clotting cascade to cause massive blood loss (Koh et al., 2006; Casewell et al., 2013). A snake's venom cocktail can include serine proteases, short toxin peptides, L-amino oxidases, disintegrins, nerve growth factors, phospholipase A₂, and snake venom metalloproteinases (SVMPs) (Casewell et al., 2013; Gutierrez et al., 2016). The nonprotein fraction of snake venom can include lipids, nucleosides, and carbohydrates, as well as metal ions (Bieber, 1979).

Though metal ions make up a minor fraction of venom, several major venom components depend on them. For example, metal ions increase the structural stability of anticoagulation factor II (Xu et al., 2002). They also act as switches to alter the function of multicatalytic enzymes (Xu et al., 2010). Several snake venom enzymes are especially dependent on divalent metal ions (Francis et al., 1992), including Ca²⁺-dependent phospholipase A₂, divalent metal ion-dependent 5′-nucleotidase, Mg²⁺-dependent phosphodiesterase, Mg²⁺- or Ca²⁺-dependent alkaline phosphatase, and Zn²⁺-dependent proteinase (Xu et al., 2002; Francis et al., 1992). Finally, metal ions are crucial components of metalloproteinases, which cause proteolytic degradation of the extracellular matrix to affect inflammation, hemorrhage, and other physiological responses in their prey (Teixeira et al., 2005).

Fortunately, several methods are available to quantify a sample's metal ion content (Maret, 2018). Of these, inductively coupled plasma mass spectrometry (ICP-MS) has several advantages (Wilschefski and Baxter, 2019). It has a large analytical range, a very low detection limit, is high throughput, and can quantify many elements in a single analysis. In this study we used ICP-MS to quantify the abundance of metal ions in venom from seven snake species found in the southern United States: the eastern cottonmouth (Agkistrodon piscivorus), the western cottonmouth (Agkistrodon piscivorus leucostoma), the broad-banded copperhead (Agkistrodon contortrix laticinctus), the southern copperhead (Agkistrodon contortrix), the Mojave rattlesnake (Crotalus scutulatus), the western diamondback (Crotalus atrox), and the eastern diamondback (Crotalus adamanteus). To the best of our knowledge this analytical technique, used previously in studies of insect (Kokot and Matysiak, 2008) and scorpion (Al-Asmari et al., 2016) venoms, has never been applied to whole snake venom. ICP-MS is well-suited to quantifying a venom's metal ion content, which is known to affect its proteolytic activity (Atanasov et al., 2013) and can indirectly reveal SVMP abundance. While SVMPs are present at high concentrations in the venoms of several snake species (Casewell et al., 2014), less is known about the concentration of the metal ion cofactors associated with these toxic enzymes and how they affect snake venom toxicity.