Purification And Characterization Of Phospholipase A₂ From Snake Venom (Trimeresurus Albolabris)

Trimeresurus albolabris, a member of the Trimeresurus, Crotalinate Viperidae, as the venomous snake, distribute mainly in Chongqing, Yunnan, Sichuan, Guizhou, Fujian, Hainan, Guangdong, Guangxi and Taiwan etc. The snake venom contains many kinds of components which can affect the blood system. In the components, Phospholipase A₂ was extensively studied.Phospholipase A₂ is rich in venom of snake and mammalian pancreas. Phospholipase A₂ specifically catalyses the 3-Sn-glycerol phospholipids of sn-2 ester bond of glycerophospholipids, leading to the generation of glycerophospolipids and fatty acids. It showed a variety of pharmacological properties, such as blood toxicity, hemolytic activity, muscle toxicity, the role of anticoagulation and induced edema. The contrast between the speciality of their structure and the complexity of their activities makes phospholipase A₂ in the snake venoms be an important model for the study of the structure-function relationship of proteins. A number of studies aimed at characterizing phospholipase A₂ of Crotalidae snakes, and numerous studies on phospholipase A₂ from the venoms of snake such as Agkistrodon acutus, kistrodon halys Pallas, T. Stejnegeri, T. mucrosquamatus have been reported, but the studies on phospholipase A₂ from the venom of snake (T. albolabris ) have been not yet reported. In this work, from the venom of snake (T. albolabris) in China we have isolated and purified a phospholipase A₂, and to futher study its properties. The main research works are as follows:1. Using three-step chromatography (CM-Sephadex C-50 ion-exchange chromatography, Sephadex G-75 gel chromatography and Q-Sepharose FF ion-exchange chromatography), we isolated and purified a phospholipase A₂ from T. albolabris venom, which was 17.2 KDa. SDS-PAGE results showed that under the reducing and non-reducing conditions, it presented only one band, and indicated that it possiblely contained a single peptide chain.2. The properties of phospholipase A₂ were studied. Its specific activity was 4.625×10⁴ U/mg as a substrate of hen's egg-yolk, and its optimum temperature was 50℃, its optimum pH was pH 8.0. The enzyme has a good thermal stability; Ca²⁺,K⁺,Na⁺ inhanced its activity, Mg²⁺,Zn²⁺,Cu²⁺,Fe²⁺ significantly inhibited its activity; EDTA and EGTA could inhibit its hydrolytic activity, DTT,PMSF andβ-mercaptoethanol weakly inhibited its activity; and it has no proteolytic activity,arginine ester hydrolysing activity and thrombin-like enzyme activty; but has some anti-trypsin activity and indirect hemolytic activity; animal experiments show that it has no edema activity.