Purification and Partial Characterization of a Coagulant Tle
Received: September 4, 2008 J Venom Anim Toxins incl Trop Dis. Accepted: February 13, 2009 V.15, n.3, p.411-423, 2009. Abstract published online: March 5, 2009 Original paper. Full paper published online: August 31, 2009 ISSN 1678-9199. PURIFICATION AND PARTIAL CHARACTERIZATION OF A COAGULANT SERINE PROTEASE FROM THE VENOM OF THE IRANIAN SNAKE Agkistrodon halys Ghorbanpur M (1), Zare Mirakabadi A (2), Zokaee F (1), Zolfagarrian H (2), Rabiei H (2) (1) Chemical Engineering Department, Amirkabir University, Tehran, Iran; (2) Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute, Karaj, Iran. ABSTRACT: Agkistrodon halys is one of several dangerous snake species in Iran. Among the most important signs and symptoms in patients envenomated by this snake is disseminated intravascular coagulation. A thrombin-like enzyme, called AH143, was isolated from Agkistrodon halys venom by gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a DEAE-Sepharose and high performance liquid chromatography (HPLC) on a C18 column. In the final stage of purification, 0.82 mg of purified enzyme was obtained from 182.5 mg of venom. The purified enzyme showed a single protein band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), under reducing conditions, and its molecular mass was found to be about 30 kDa. AH143 revealed clotting activity in human plasma, which was not inhibited by EDTA or heparin. This enzyme still demonstrated coagulation activity when exposed to variations in temperature and pH ranging, respectively, from 30 to 40°C and from 7.0 to 8.0.
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