Purification And Characterization Of The Disintegrin From Gloydius Brevicaudus Venom

Disintegrin which contains the amino acid sequence Arg-Gly-Asp(RGD) orLys-Gly-Asp(KGD),has been implicated as a recognition site in interactions betweenintegrin and its ligands.It exhibit a wide array of biological activities, such as inhibitplatelet aggregation, angiogenesis and tumor migration.This study was designed toisolate and purify disintegrin from Gloydius brevicaudus venom and analyse itscharacterization and biological activities.1. Purification and characterization of the disintegrin from Gloydius brevicaudusVenom(GBV)Six fractions(Ⅰ～Ⅵ) were isolated from GBV by Gel filtration chromatography onSuperdex 75 .The fraction â…£inhibited the platelet aggregations induced by ADP withmolecular weight lower than 10000 dalton,so it suggested as a contained disintegrincomponent.A disintegrin named Gbvâ…£4 was purified from the fraction by sephadexG-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18reverse chromatography. It was homogeneous as a single band showed onSDS-polyacrylmide gel electrophoresis (SDS-PAGE,Tris-Tricine system) withmolecular weight 8746 dalton as calculated by Image Master VDS system.Theisoelectric point of Gbvâ…£4 was pH 5.2 by disc polyacrylamide gel eletrophoresis.Gbvâ…£4 exhibited no detectable phospholipase A₂(PLA₂)activity with the pH-stattechnique and proteinase activity according to the method of Rick.2.The effects of Gbvâ…£4 on the human platelet aggregation.According to the Born's method,the effects of Gbvâ…£4 on platelet aggregationinduced by ADP and thrombin were assayed.The results showed that Gbvâ…£4dose-dependently inhibited ADP-and thrombin-induced platelet aggregation with IC₅₀0.22 and 1.327μg/ml respectively.3.The effect of Gbvâ…£4 on the proliferation of human gastric carcinoma SGC-7901cellsThe effects of Gbvâ…£4 on cultured SGC-7901 cells was observed by SRB(Sulforhodamine B)method.The results showed that Gbvâ…£4 inhibited proliferation ofSGC-7901 cells and the inhibited effect was dose dependent, but it did't be found theincreased effect following delayed time. The IC50(24h) value was 71.68μg/ml.4.The effect of Gbvâ…£4 on the proliferation of human umbilical vein ECV304 cells The inhibition of Gbvâ…£4 on cultured ECV304 cells was observed and IC50 valuewas assayed by SRB method.The result showed that Gbvâ…£4 inhibited theproliferation of ECV304 cells in a dose-dependent manner with IC50(24h)25.42μg/ml.Furthermore,The ECV304 cells undergo remarkable morphologicalchange by treatment with Gbvâ…£4:the cells retracted ,turn to ground,formed gaps andmulticelluar aggregation.5.The effect of Gbvâ…£4 on angiogenesis of chick chorioallantoic membrane(CAM) The antiangiogenesis effect in vivo of Gbvâ…£4 on CAM assay was observed and thethe ratio of area of blood vessel and area of CAM in measure area (VA/CAM) wasused to asses the inhibition.The result showed that Gbvâ…£4(3.098,11.79μg)inhibitedthe angiogenesis in vivo: the morphology of blood vessels on CAM were basicallynormal,but the blood vessels distributed scarcely .The VA/CAM was less than controlgroup significantly.Conclusions A disintegrin Gbvâ…£4 was purified from Gloydius brevicaudus venom withmolecular weight 8746 dalton,PI 5.2. It was a potent platelet aggregation inhibitor.Gbvâ…£4 inhibited proliferation of cultured tumor cells SGC-7901 ,human umbilicalvein endothelial cells and antiangiogenesis of CAM.