Abstract
Taiwan Cobra (Naja atra) is distributed based on eastern and western regions. Eastern cobra (bCTXs) is larger in size and fully black on its belly, whereas western cobra (wCTXs) is smaller and belly is white or gray. In the previous study from our lab, we found that east and west cobra cardiotoxin also have regional difference. West cobra venom contains more of cardiotoxin (CTX) A2 and A4, but lacks A6. Anionic citrate is one of a major component of cobra venom and around 50 mM citrate is present in Taiwan cobra venom. In cobra venom, effect of citrate on toxicity is poorly understood than inhibitory effect on cation-dependence action from venom toxins. In previous study, a citrate ion bound to Lys-23 and Lys-31 near tip of loop II stabilizes CTX-A3 homodimer and hydrophobic contact of functionally important loop I and II regions. Sequence alignment revels that both Lys-23 and Lys-31 are conserved residues in A3 and A6 (bCTXs) but not for A2 and A4 (wCTXs). The interaction of glycosaminoglycan and CTX in citrate might play an important role in cell membrane retention for bCTXs than wCTXs. Our results shows that bCTXs is about six times stronger than wCTXs in retention of heparin surface when added citrate in surface plasmon resonance(SPR), indicating that Lys-23 and Lys-31 have an impact for retention. From SDS-PAGE, we observed that adding cross-linking reagent and citrate induced heterodimer in CTX A3-A6 which was more evident than CTX A2-A3 and CTX A4-A3 heterodimer. Further retention of heparin surface in bCTXs and wCTXs are analyzed from reverse phase high performance liquid chromatography and we found that A3 had most significant retention in all CTXs. In cell viability assay, wCTXs is more cytotoxicity than bCTXs and citrate group have higher cell death than non-citrate group. On the other hand, increase in citrate concentration leads to improve in cell death. These results suggest that citrate is involved significantly to induce the formation of dimerization in bCTXs which can lead to more cell death. The dimerization in cardiotoxins might suggest relationships with cell death. We also found wCTXs and bCTXs cell death mechanism is completely different.