Acute toxicity of vipoxin and its components: is the acidic component an “inhibitor” of PLA(2) toxicity?

Vipoxin is a heterodimeric neurotoxin isolated from the venom of the Bulgarian long-nosed viper Vipera ammodytes meridionalis. Vipoxin represents a noncovalent association of two subunits – a basic and toxic phospholipase A(2) enzyme, and an acidic non-enzymatic component (vipoxin's acidic component). It was postulated that the phospholipase A(2) subunit was more toxic than the whole vipoxin complex and the function of the acidic component was to reduce the enzymatic and toxic activities of the basic phospholipase A(2). In the present study, we report new data on the acute toxicity (LD(50)) of vipoxin and its individual separated components. Vipoxin LD(50) (mice, i.p. and i.v.) values were found to be 0.7–1.2 mg/kg b.w. (i.p.) and 0.9–1.3 mg/kg b.w. (i.v.). The established LD(50) values for the separated pure phospholipase A(2) subunit are higher – 10.0–13.0 mg/kg b.w (i.p.) and 2.2–3.0 mg/kg b.w. (i.v.), i.e. the individual phospholipase A(2) subunit displays less toxic activity than vipoxin, contrary to the data published in the literature. The reconstituted vipoxin complex (obtained after preliminary incubation of pure separated phospholipase A(2) and acidic component showed enzyme activity and toxicity comparable to that of the native vipoxin complex. Addition of acidic component to the phospholipase A(2) subunit showed a positive effect on the enzymatic activity, reaching maximal enzyme reaction rate of acidic component to phospholipase A(2) molar ratio of 0.8:1 on using 4-nitro-3-octanoyloxy-benzoic acid as substrate. For the first time we showed that the acidic subunit was absolutely required for the toxic activity of vipoxin. Based on the obtained results, we assume that the function of the acidic component is to stabilize the neurotoxin's quaternary structure, required for its toxic and enzymatic activities, similarly to the role of the acidic component of crotoxin.