Isolation and purification of toxic fractions from the venom of Mesobuthus eupeus

Background: Mesobuthus eupeus of the buthidae scorpion family is a native species in Iran (especially in Khuzestan region). The venom of scorpions, particularly buthidae family, is mainly composed of various bioactive components including various toxins. These toxins affect different ionic channels in excitable membranes of insects or mammals. In this study, the toxic fractions of Mesobuthus eupeus crude venom were isolated and those with higher toxicity were purified. Methods: Six toxic fractions from the crude venom of Mesobuthus eupeus were separated by gel filtration chromatography on sephadex G-50 in the equilibration with 20 mM ammonium acetate buffer, pH 4.7. Then the most toxic fractions were separated by cation exchange column chromatography and their purity was confirmed by electrophoresis. Results: The amount of 816 mg protein of soluble crude venom was applied on sephadex G-50 column chromatography, and 815 mg protein was yielded from the 6 separated fractions. From these fractions, fractions III and IV were toxic to mice. The LD50 values (Lethal Dose 50) of them were 0.40 and 0.46 mg/kg in mice, respectively. Fraction III was separated into 20 distinct subfractions. Through examining the toxicity of those subfractions to the mice, the toxic effect of subfractions III.14 to III.19 on mice was detected. From 250 mg protein of fraction III, applied on CM-sephadexC-25, 242.75 mg (97%) protein was attained in the separated subfractions. Conclusion: Three distinct toxins were obtained in pure form from the venom of Mesobuthus eupeus. The subfraction III.14.4 with an LD50 of 0.11 mg/kg in mice had the highest toxicity. The structural characteristics, physiological and pathological effects of these three purified toxin subfractions can be studied in the future studies.