ABSTRACT
In the present study we evaluated the effect of secreted phospholipase A2 (sPLA2) (the toxic subunit of the heterodimeric neurotoxin vipoxin, isolated from the Bulgarian long-nosed viper Vipera ammodytes meridionalis) on hemolysis, erythrocyte morphology and platelet aggregation. Hemolytic activity of sPLA2 was examined in the presence of saturated (palmitic) and unsaturated (oleic) fatty acids and it was found that oleic acid increased the hemolytic activity of sPLA2 in a concentration-dependent manner, compared to the effect of palmitic acid and controls. The addition of heparin to red blood cells (RBC) suspension containing sPLA2 or mixture of sPLA2 and the corresponding fatty acid led to an inhibition of hemolytic activity. The effect of sPLA2 on RBC morphology resulted in formation of echinocytes (spherocyte subtype), suggesting that RBC could be the possible targets attacked by sPLA2. Vipoxin sPLA2 inhibited (in a dose-dependent manner) platelet aggregation when arachidonic acid and collagen were used as inducers, while in the case of ADP its inhibitory effect was inappreciable.
ABSTRACT
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 A2 enzyme, and an acidic non-enzymatic component (vipoxin's acidic component). It was postulated that the phospholipase A2 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 A2. In the present study, we report new data on the acute toxicity (LD50) of vipoxin and its individual separated components. Vipoxin LD50 (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 LD50 values for the separated pure phospholipase A2 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 A2 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 A2 and acidic component showed enzyme activity and toxicity comparable to that of the native vipoxin complex. Addition of acidic component to the phospholipase A2 subunit showed a positive effect on the enzymatic activity, reaching maximal enzyme reaction rate of acidic component to phospholipase A2 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.
