Purification And Characterization Of A Fibrin(ogen)olytic Enzyme From The Venom Of Agkistrodon Haly Pallas

Objective: To purify fibrin(ogen)olytic enzymes from the venom of Agkistrodon haly Pallas and study the characters. Method: Filtration and anion-exchange chromatography to purify fibrin(ogen)olytic enzymes; Fibrin plate method to determine fibrino- lytic activity; Fibrin overlay after PAGE for localization of fibrinolytic activity ;SDS-PAGE to determine molecular weight; IEF to determine isoelectric point.Fibrin(ogen) degradation was demonstrated on a SDS- PAGE. Platelet aggregation assays were performed in human platelet- rich plasma.Chromogenic activity assays with S-225 land S-2238 to in- vestigate substrate specificity and determine Km on S-2251 and S-2238. Result: Fibrin overlaying after PAGE make sure that there are at least four different mobility fibrinolytic principle in Agkistrodon halys pallas venom.A fibrin(ogen)olytic enzymes was isolated and purified from Agkistrodon ha/v Pallas .The molecular weight was estimated as 23.8k i-i by SDS-polyacrylamide gel electrophoresis. The isoelectric point was found to be pH4.87. The enzyme retains fibrinolytie activity and not changes structure although incubated at 560C for 30 mins. The fibrino- lytic activity was inhibited by EDTA and f3 -mereaptoethanol with the structure changing suggesting the enzyme is a metalloprotease and the disulfide bonds are necessary for holding the structure. The activity was also inhibited by Cu2~,Co2~ and unaffected by PMSF,PABA,Mg2~, Zn2~,Ca2~ and K~. When fibrinogen was incubated with the enzyme, the A a chains was hydrolyzed faster than the B ~ chains , but the Y chains was unaffected.It not only hydrolyzed fibrinogen but also acted on fibrin. The enzyme readily hydrolyzed a chains ,slowly hydrolyzed ~ chains and unaffected v chains . The enzyme can be classified as a - fibrin(ogen)ase . It is a nonhemorragic metalloprotease.The enzyme in- hibit platelet aggregation induced by ADP .The Km of the enzyme on S- 2251 is l.72X 104mo1/L and S-2238 is 1.88X 104mo1/L.N-terminus of FLE are :QRFPTRYIEL.