Anti-hemorrhagic Activity of Wild Custard Apple (Annona senegalensis) Ethanolic Leaf Extract on Spitting Cobra (Naja negricollis) Metalloprotease.

The main feature of Naja nigricollis venom is its high proteolytic activity responsible for most of local and systemic effects observed during envenomation by this snake. In this present work we reported the purification and characteristics metalloprotease from Naja nigricollis venom by combination of two step chromatography; a gel filtration (sephadexG-75) and an ion – exchange(DEAE–sephadex –A-50). Ethanolic extraction of the leaf of Annona senegalensis was carried out. An hemorrhagic Naja nigricollis metalloprotease(NNMP) was partially purified from Naja negricolis venom using sephadex G-75 and DEAE-sephadex A-50 column chromatography. Purification of the hemmoraghic metalloprotease was about 11-fold with a total recovery of 5.8% from the crude Naja negricollis venom. Biochemical characterization of NNMP show a Km of 10.5M and Vmax  of 45.5 unitsmg-1. The hemorrhagin show optimal stability at PH 5.5 acetate buffer and thermally stable at 400C. NNMP has activation energy as 0.25kjmol-1 and it caused hemorrhage when injected intra-dermally in albino mice suggesting hemorrghic metalloprotease. NNMP hemorrhagic activities were highly inhibited by ethylenediaminetetraacetic acid (EDTA) and the ethanolic leaf extract of Annona senegalensis.