Studies On Seperation, Purification And Anti-Tumor Activity Of Snake Venom

The distribution of snakes in the world is quite extensive and the numbers of their species is very numerous, and there are more than 200 species and 54 subspecies of snakes have been found in China mainland. Snake venoms contain complex mixtures of hundreds of biologically active substances and the venom components are very useful for pharmaceutical applications. So the study and development of the bioactive components in snake venom has become an important research area in basic medicine and pharmaceutical industry. Some components in snake venoms such as enzymes and neurotoxins have been purified and their biological activities applied. However, many protein components in snake venoms and their biological functions are still undetermined. Comparing with the international research development, only a few crude venoms or partially purified protein components of their venoms have been studied in China, and the anti-tumor components in majority of the venoms have not been identified yet. Here, we take the naja naja atra venom, which is abundant in Guangxi, South China, as the source of samples in this paper, performed the purification and seperation of the snake venom, and made the screening and evaluation of anti-tumor activity of these protein components, for the purpose of the discovery of bioactive agents with potential applications in clinical therapy.First, we take the venoms of Naja naja atra, Agkistrodon halys, Bungarus fasciatus and Vipera russelli as the source of samples, and made the screening of anti-tumor activity of these four snake venoms, anti-tumor activity showed as following trend:naja naja atra＞Agkistrodon halys＞Bungarus fasciatus＞Vipera russelli. MTT assay revealed that the half maximal inhibitory concentration (IC50) of naja naja atra venom and Agkistrodon halys venom towards HepG2 cells were 5.12 and 22.50μg/mL, respectively; While the IC50 of Bungarus fasciatus venom and Vipera russelli venom towards HepG2 cells were all more than 100μg/mL.Naja naja atra venom was then selected for further research for its significant anti-tumor effect. By using FOCUSTM-Mammalian Proteome kit and SpeedVac concentrator processing, naja naja atra venom crude purified protein was prepared. MTT assay revealed that the IC50 of naja naja atra venom crude purified protein towards HepG2 cells was 1.07μg/mL. By comparing IC50 values, both naja naja atra venom crude purified protein and naja naja atra venom were found to show less toxic towards human normal hepatocyte HL-7702 line than tumor cell line tested, while the former one possessed a more selectivity.Cell growth curve indicated that naja naja atra venom crude purified protein significantly inhibited the proliferation of HepG2 cells in a concentration-and time-dependent manner. Using acridine orange (AO) staining under fluorescence microscope, we observed the consistent result. In cultures without naja naja atra venom crude purified protein, HepG2 cells grow in monolayer, cells distribute evenly, are fusiform in form, and grow normally. While cells treated with low dose naja naja atra venom crude purified protein, the cells begin to shrink, and vacuoles appear between the cells. As concetration goes on, cells showed typical apoptosis features characterized homogeneously Kelly fluorescence by volume reduction, chromoatin condensation, nulear fragmentation with dense Kelly flurescence stain and appearance of apoptotic bodies.An ion-exchange column of DEAE-sephacel was employed for the further separation of the 16 mg naja naja atra venom crude purified protein. A convex gradient was then applied up to 0.5 M Sodium chloride Tris-HCl buffer, pH 8.0 at a flow rate of 0.5 mL/min. The protein absorption was monitored at 280 nm. There are four absorbance peaks in the ion-exchange elution profile, the first and second peak were major components of the venom crude purified protein. Comparative study of reverse-phase chromatographic elution profiles from each fraction was carried out. Two mobile phases, A (0.1%v/v TFA,98%v/v ACN) and B (0.1%v/v TFA,2%v/v ACN), were used for 20μL filtered samples. A linear gradient of 14.7%ACN to 88.2%ACN (15-90%mobile phase A) in 105 min running time at a flow rate at 0.8 mL/min was used. The first eluted peak between fractions 9 and 12 contained a variety of proteins, of which two types of high-level protein mixture, that differ in term of hydrophobic nature and ultraviolet absorption characteristic; and chromatography indicated three single protein play major role in the each following absorbance peaks, each protein substance vary on hydrophobicity and spectral characteristic. The protein concentration of the 40 fractions were determinated with Bicinchoninic acid method, Bovine serum albumin was used to make a standard curve. The results were consistent with ion exchange elution pattern. We selected representative fractions corrpnding to each eluted peak based on previous analysis, and then made the follow-up screening and evaluation of the anti-tumor activity. The molecular weight was determined by SDS-PAGE method. Results show the IC50 values of selected fraction were relatively higher than corresponding value of naja naja atra venom crude purified protein. The first peak in ion-exchange elution profile remained an intense anti-tumor activity, the main venom component of this peak had a molecular weigh range between＜14 and 30 KDa. The protein composition of the naja naja atra venom crude purified protein fractions were analyzed by using in-solution digestion,2D-LC, QStar Elite MS/MS. Characterization of the related protein subtance is still in progress.