Isolation, Purification and Characterization of Hyaluronidase and Phospholipase A2 Enzymes of Echis ocellatus and Naja nigricollis Snake Venoms

Abstract

Issues related to snakebite has for long been of high economic and medical importance. Management and treatment of snake envenomation has always been an issue, especially in remote areas, where anti-snake venom and facilities for its storage are not available, coupled with the high specificity and instability of anti-venom. Venom enzymes are usually responsible for so many tissue necrotic activity after bite. This research was aimed at isolation, purification and characterization of Hyaluronidase and Phospholipase A₂ (PLA2) Enzymes of Echis ocellatus and Naja nigricollis snake venoms. Isolation and purification of these enzymes were done using a two-step process which included gel filtration on Sephadex G-75, active fractions were applied to ion–exchange chromatography on DEAE (Diethylaminoethyl) cellulose. Individual active fractions of each venom, was then subjected to SDS–PAGE for molecular weight extrapolation. Enzyme characterization was done on the two isolated enzymes for the two snake venoms used. N. nigricollis enzymes were revealed to have an optimum temperature of 37 ^oC and 55^oC, while that of E. ocellatus had 37 ^oC and 40^oC, with a pH of 3.5 and 8.0 for both the venom enzymes. Velocity kinetics carried out shows that N. nigricollis PLA₂ has the highest V_max value of 30.567mg/min, while E. ocellatus PLA₂ however has the highest K_m value of 4.5378mg/ml. Purification and characterization done in this research has revealed/confirmed that these venoms contain Hyaluronidase and PLA₂ enzymes, giving a better understanding of the enzymes which will aid in the management and treatment of snake envenomation from these snakes.