Affinity-purification of fibrinogenase with high proteolytic activity from Agkistrodon halys (Chinese) Venom.
To purify and characterize the fibrinogenase with high proteolytic activity from Agkistrodon halys (Chinese) Venom. Monoclonal antibodies against fibrinogenase were prepared and a novel affinity chromatography equipped with a monoclonal antibody against fibrinogenase was developed and applied for the purification of fibrinogenases. The purified fibrinogenase was identified by fibrinolytic activity assay, and antithrombosis activity assay. HPLC chromatography and SDS-PAGE analysis demonstrated the uniformity and purity of the purified fibrinogenase. In comparison with a conventional A-50 chromatography method, affinity-purified fibrinogenase showed higher activity (3631 U mg(-1) vs 501 U mg(-1)). In addition, the physiological activity of the fibrinogenase both in vitro and ex vivo showed the purified fibrinogenase can specifically degrade beta-, gamma-fibrinogen and has a high anti-thrombotic activity. In conclusion, the purified fibrinogenase by affinity column were shown to be homogeneous and showed a high and specific proteolytic activity against beta-chains of fibrinogen molecules and antithrombosis activity.
Ma
Biao
B
College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100083, China.
Zhang
Ying
Y
Wu
Dan
D
Jia
Jianping
J
Xu
Wentao
W
Luo
Yunbo
Y
D017836:Agkistrodon; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003636:DEAE-Cellulose; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D007120:Immunochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D014757:Viper Venoms / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D014757:Viper Venoms; D003636:DEAE-Cellulose; D012697:Serine Endopeptidases; C412879:venombin B