Isolation and enzymatic characterization of the snake venom hyaluronidase from Naja melanoleuca (Forest cobra) venom

Abstract

Hyaluronidase is ubiquitous enzyme in snake venoms components, known originally as “spreading factor” because they cleave hyaluronan found in the extracellular matrix (E.C.M) of connective tissue, facilitating toxin diffusion into the tissue and blood circulation of the prey/victims. The purification procedure of a hyaluronidase from Naja melanoleuca venom is described. It involves basically size exclusion chromatography on Sephadex G-75 at PH 6.0, followed by re-chromatography of active fractions on ion exchange chromatography using DEAE-cellulose. The specific activity of purified enzyme was 0.25 TRU/mg against 0.03 TRU/mg for the crude venom, representing 8.62 purification fold. The purified hyaluronidase has molecular weight of approximately 54 KDa on SDS-PAGE. The enzyme had optimal pH 6 and temperature 38 °C respectively. Its KM was found to be 0.381×10-3 mg/min at 38 °C. This research showed that N. melanoleuca hyaluronidase exhibited high affinity for hyaluronic acid.