Purification and Characterization of the Antibacterial Component from Taiwan Cobra (Naja naja atra) Venom

• Main Authors: Wu Chia Hann, 吳佳翰
• Other Authors: Wu Wen Guey
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/96323285329564810860

碩士 === 國立清華大學 === 生命科學系 === 89 === ABSTRACT Cardiotoxins (CTXs) from cobra venom cause some toxic effects, such as cytolysis. However, the exact mechanism of lytic effects caused by CTXs remains unclear. Besides, like many cationic bactericidal peptides, CTXs are also polycationic. This makes we wonder if they are antibacterial? If they are, by what kind of mechanism? Besides, in snakebite victims, the bitten tissues would be destroyed and followed by cell death (necrosis) if not well treated. Some people think this may be due to bacterial infection. However, CTXs also cause necrosis. Therefore, if CTXs or other components in snake venoms are antibacterial, then the necrosis might be caused by CTXs rather than bacterial infection because of the inhibition of the bacterial growth on the bitten tissue. Thus, the arm of this study is to find out if snake venoms are antibacterial. Besides, we will try to purify the antibacterial component and characterize it. My results showed crude N.n.atra venom did show antibacterial activity against both gram positive and negative bacteria. However, we didn’t see CTXs exhibited any antibacterial activity against S. aureus. Therefore, SP-Sephadex C 25, Sepharose CL-6B, phenyl 5PW, and Superdex 75 chromatography were used to purify the antibacterial component. To our surprise, the antibacterial fractions of each column chromatography also showed L-amino acid oxidase (LAO) activity. We started with one-gram crude venom but ended up getting approximate one-milligram antibacterial component after purification via Superdex 75 column. Because LAO was a homodimeric protein with approximate molecular mass of 120 kDa, we were supposed to see a 60-kDa band on SDS-PAGE. However, the purified compound exhibited both 120-kDa and 60-kDa bands on reduced SDS-PAGE instead of a 60-kDa band. Two peaks derived from RP-HPLC showed individual single band of 120 kDa and 60 kDa, respectively. It indicated the 120-kDa band was due to impurity. Besides, catalase at high concentration inhibited the antibacterial activity of the purified compound. This meant the purified compound exerted its antibacterial activity by producing hydrogen peroxide. All signs showed the exact antibacterial component might be LAO. Because of the insufficient number of bacterial strains used, the fact that the necrosis might be caused by CTXs rather than bacterial infection needs to be confirmed in the future.