Purification and Biochemical Characterization of Hyaluronidase from Egyptian Echis pyramidum pyramidum Venom

Abstract

A novel hyaluronidase was purified to apparent homogeneity from Egyptian Echis pyramidum pyramidum venom. The enzyme was purified 7.6-fold by Sephacryl S-200 size exclusion followed by CM-Sepharose cation-exchange chromatography with a specific activity of 610 U/mg over the activity of whole venom (80 U/mg). The molecular mass of the purified enzyme was approximately 48 kDa in its native and reduced forms. The maximum activity was estimated at pH 5.5 and 40 o C. The purified hyaluronidase has an absolute activity towards hyaluronic acid with Km and Vmax values of 20µg/ml and 40µg/min, respectively. The Na + ion enhanced the hyaluronidase activity, whereas Fe +2 , Ni +2 , Cu +2 , Co +2 , Zn +2 , Mn +2 , o-phenanthroline (5 mM) and heparin (10 units) inhibited the enzyme activity by 100%. Antisera against the venoms of Echis pyramidum, Echis coloratus, Cerastes cerastes, Cerastes vipera and Naja haje greatly neutralized E. pyramidum hyaluronidase to a varied extent. In contrast, antisera against Naja nigricollis negatively neutralized the hyaluronidase activity. The study concluded that the purified enzyme shared similar immunogenic properties with venoms of vipers rather than elapids. It also demonstrated the importance of characterization of enzymes from venoms of different species to explore the venomics and antivenomics of Egyptian snake species.