Purification characterization and inhibition studies on Phospholipase C from Opisthacanthus capensis (Black Scorpion) venom

Abstract

Phospholipase C from Opisthacanthus capensis venom was partially purified and characterized. The enzyme was purified 105.2 fold with an overall yield of 13% using various purification steps involving ammonium sulphate precipitation, protamine sulphate treatment, sephadex G-75  fractionation and DE-52 anion exchange chromatography. The purified enzyme was  homogeneous with a molecular weight of 29 kDa. The  phospholipase C has pH and temperature optima of 7.2 and 600C, respectively with activation energy of 25KJ/mol and t1/2 of 1.50 hr. Initial velocity studies on O. capensis venom phospholipase C revealed a KM of 0.02 mM and Vmax of 0.015 μmol/min. Studies on the effect of pH on KM and Vmax gave PKa1 of 6.9 and PKa2 of 7.4 with enthalpy of ionization of 20 KJ/mol suggesting  histidine in the active site. The enzyme was positively modulated by Mg2+, Zn2+ and Ca2+ and negatively by Fe2+. While Hg2+ produced complete inhibition. Various concentrations of leaf aqueous extract of Momordica charantia also inhibited the activity of O. capensis venom phospholipase C in vitro with a competitive pattern. This study revealed the presence of phospholipase C in O. capensis venom and gave some scientific basis for the use of the
plant in the treatment of scorpion envenomation.

Keywords: Phospholipase C; Opisthacanthus capensis;  Momordica charantia; Venom