Rurification,Characterization And Gene Cloning Of An Acidic Phospholipase A₂ From Guangxi Ophiophagus Hannah(King Cobra)Venom

An acidic phospholipase A2 ,designated as APLA2-1, was isolated and purified from Guangxi Ophiophagus hannah( king cobra) venom by successive chromatography on Sephadex G-75, CM-Sepharose CL-6B and DEAE-Sephadex A50 column. It had relative molecular weights of 15 ku and p1 of 5.4. It had a higher phospholipase A2 activity(108 μmol/mg·min-1) . The amino composition analysis showed that it had seven pairs of bisulfide bond and the acidic amino(Asp,Glu) occupied a larger proportion in its molecular. Its N-terminal sequence was NLIQFGNMIQCTVPGF determined by direct protein sequencing. It put up lethal activity (LD50-6.6 mg/kg) to mouse. It also exhibited indirect haemolytic , inhibit platelet aggregation, myonecrotic, cardiotoxic and very weak cytotoxic activities. In order to attain the complete protein sequence of APLA2-1, this study had also determined the eDNA sequence by gene clone method and therefore deduced the protein sequence. The open reading frame(ORF) of APLA2-1 consisted of 453 bp encoding 151 amino acids, which included a signal peptide of 27 amino acids and a mature peptide of 124 amino acids, possessed the typical characteristic of PLA2. Its F-iS amino sequence in N-terminal was identical to the sequence determined by direct protein sequencing. Its secondaiy structure and partial physic-chemistiy character were predicated by Antheprot 4.3 c software. Homology analysis indicated that the APLA2-1 had very high homology with PLA2 from Fujian and Taiwan king cobra venom and showed lower homology with some acidic PLA2 from cobra venom. The gene cloning and characterization research of APLA2-1 would provided a good basis for further discussing the relation of structure and function , and the studying of evolution of PLA2 from snake venom.