Purification And Characterization Of Metalloproteinase From Naja Atra Venom

Snake venom is one of the most abundant natural proteinase mixture, has attracted the attention of researchers for long time. Snake metalloproteinase belongs to acidic proteinase family; the latter is widely present in almost all specias, from simple bacteria to human body. Metalloproteinase is one of the main functional of components in snake venom. Divalent cations, such as Zn2+and Ca2+, are required to maintain the activity and structural integrity of the enzymes. Snake venom metalloproteinases can inhibit platelet aggregation, and increase the permeability of capillary vessel. As a consequence, acted with other factors, e.g. thrombin-like enzymes, the metalloproteinase can cause bleeding. As a potential platelet aggregation inhibitor with anticancer activities, further research of metalloproteinase in Chinese cobra venom is necessary.By using ion exchange and gel filtration chromatography, followed by Sephadex C-25 chromatography, Sephadex A-25 chromatography, and Sephadex G-75 chromatography, an acid protease was isolated and purified from the Chinese cobra (Naja atra) venom. Results showed that the protease is a metalloproteinase and its abundance is about 0.1%. With casein as substrate, PPase protein hydrolysis enzyme specific activity of the metalloproteinase was 0.0967 U/mg. Ca2+, Cu2+and Zn2+show activating effect on the enzyme activity; while Fe3+,β-mercaptoethanol and EDTA show inhibitory effects. The effect of Mg2+on the enzyme is not obvious; but K+and Na+show relatively low inhibitory effect on the activity of the enzyme, tested with the casein as substrate. SDS-PAGE of the enzyme protein showed a band, the molecular weight of the enzyme is about 71.0 kDa. Compared with data reported in literatures, the metalloproteinase is supposed to be a novel protease.MTT assay was applied to test the effect of the metalloproteinase on Hela and PC12 cell lines. Present results suggested the metalloproteinase has anti-tumor activity. It showed activity to hydrolyze extracellular matrix of tumor cells in vitro. Further studied of the anticancer activity the metalloproteinase will be valuable.