Ammodytagin, a hemorrhagic Zn-dependent metalloproteinase from Vipera ammodytes ammodytes (Vaa) venom, is a glycosylated heterodimer of 108 kDa, as determined by MALDI mass spectrometry. Partial amino acid sequencing by Edman degradation and MS/MS analysis identified sequences belonging to metalloproteinase, disintegrin-like and cysteine-rich domains, which in addition to its heterodimeric nature allows classification into the P-IIIc group of snake venom metalloproteinases (SVMPs). Only few members of that group have been described so far. Ammodytagin possesses potent azocaseinolytic activity which can be inhibited by EDTA, Zn2+ and DTT. It cleaves insulin B-chain, hydrolysing it at positions Gln4–His5, His10–Leu11 and Tyr16–Leu17. Furthermore, ammodytagin acts as a strong hemorrhagin in both rats and mice. Investigation of a substrate specificity revealed that the hemorrhagic activity of the novel SVMP might be the result of its involvement in cleavage of basal membrane components and depletion of fibrinogen, prothrombin and factor X in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective Vaa specific antivenom should be directed.
COBISS.SI-ID: 25082151
In this work we prepared rabbit antisera containing functional antibodies (Ab) specific for each class of pathology-inducing Vipera ammodytes ammodytes (Vaa) venom constituents, haemorrhagic metalloproteinases (H) and neurotoxic ammodytoxins (Atxs), separately and for both together. The involvement of these Ab in neutralising the toxicity of whole Vaa venom in mice was assessed using the ED50 assay, with the primary aim of quantifying the active compound neutralising venom-induced pathology of the antivenom. The ED50 assay in mice is the only regulatory approved assay for estimating antivenom potency. Fully functional anti-Atx Ab were shown to be responsible for neutralising the venom toxicity, while anti-H Ab are not protective in this assay. Thus, the mouse ED50 assay, intended to measure the active principle of the antivenom, does not measure Ab specific for Vaa venom haemorrhagins, and consequently does not fulfil its primary task from the regulatory point of view.