Purification Of Phospholipase A₂ From Agkistrdon Blomhoffii Ussrurensis Of Chang-bai Mountains And Studies On Its Anti-tumor Activities

Agkistrdon blomhoffii ussrurensis of Changbai Mountains, a member of the Agkistrondon blomhoffii ussurensis Emelianof, Crotalinae Viperidae, popular named as Manushi, mainly distribute in the northeast of China, Korean and parts of Russia.Phospholipase A₂(PLA₂) catalyses the hydrolysis of the sn-2 ester bond of glycerophospholipids, leading to the generation of NEFAs and lysophospholipids (lysoPLs). There are many PLA₂ isoenzymes which exist in snake venoms. The homology of amino acid sequence is above ninety percent and the structures are very similar, but they exhibit diverse pharmaco-physiological functions.11 components were obtained from the crude venom which was purified elementarily by DEAE Sephadex A-25. The result of SDS-PAGE electrophoresis showed that peak 5-2-2 which was purified by Sephadex G-75 from peak-5 having phospholipase A₂ activity was tri-component, and peak 7-3 which was purified by Sephadex G-75 from peak-7 having phospholipase A_<sub>2 activity was mono-component. Using Agilent ZORBAX 300SB- Reversed-phase C18 Chromatography to detect peak-7-3 by HPLC, it was found to be mono-component, and the ratio of impure peaks was very low, not exceeding 3%. At last we got 70mg of the PLA₂ with a protein concentration of 0.8mg/ml.Compared with the PLA₂'s specific activity which was 41μmol/mg min, the crude venom's PLA₂ activity was 17μmol/mg min, which meant that the ratio of activity recovery was over 100%. The reason of the phenomenon may be that the different components in the crude venom can compete with each other which will lead to the reduction of PLA₂ activity, as a result, the activity of crude venom is far lower than it of PLA₂ being purified. And the optimum temperature of the PLA₂ is about 50℃; the optimum pH is about pH8.5. When the PLA₂ was preserved in 100℃circumstance for 1h, the activity of the PLA₂ was not inactivity completely but with 40% of the activity still existing. It showed that the enzyme getting from the crude venom had a preferable thermal stability. And also, it can exist stably in acidic surroundings, but when pH is higher than 9,its activity disappeared rapidly.The study on anticancer activity in vitro showed that the PLA₂ has little apparent inhibition to Hela and K562 cells. According to "target site and action site" theory, if the PLA2 wants to generate pharmacological and toxicological activity, there should be binding sites on the surface of the PLA2 which can combine with target cells. Furthermore, it can combine with the target sites of the target cells. As a result, it can exert pharmacological and toxicological activity to hydrolyze cells. It is possible that there are no binding sites combining with target cells on the surface of the obtained PLA2. So it can not inhibit the growth of the cells for it can't combine with Hela and K562 cells.Simultaneously, we also studied the synergic effect of a cardiotoxin-like basic protein with a cardiotoxin from Cobra venom. A cardiotoxin(CTX) and a cardiotoxin-like basic protein(CLBP) were obtained from Cobra venom by CM-Sephadex C-25,Sephadex G-75 and HPLC chromatography. Through proliferation assay, it was noticed that the cardiotoxin-like protein exhibited synergic effect with the cardiotoxin which was at a low concentration when they were co-applied to A549 cell lines, while the synergic effect would be undetectable when CTX was at a high concentration. It is said that some cardiotoxin can form stable dimeric polymer or multipolymer when they are in the formation of crystal structure. Because of the basic character, three fingers, CLBP can simulate lethal toxin in order to kill cells, when the concentration of CTX is so low that it can kill cells. But when concentration of CTX is high to some extent, CLBP can't combine with substrate because of competition effect, thus the synergic effect disappeared.