Enzymological characterization of FII(a), a fibrinolytic enzyme from Agkistrodon acutus venom.
OBJECTIVE
To study the enzymological characterization of a fibrinolytic enzyme (FII(a)) from Agkistrodon acutus venom.


METHODS
The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FII(a) was determined by atomic absorption spectroscopy.


RESULTS
After incubation with FII(a) (0.25 g/L), Aalpha-, Bbeta- and gamma-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h , respectively. The molecular weights of major degradation products were 45,000 and 41,000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FIIa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171+/-25 mg/kg), potassium (489+/-17 mg/kg) and calcium (319+/-13 mg/kg) were found in FIIa. Zn2+, Ca2+ and Mg2+ could recover the fibrinogenolytic activity of FIIa, which was inhibited by EDTA. Only Ca2+ could recover the fibrinogenolytic activity inhibited by EGTA.


CONCLUSIONS
FIIa can degrade the Aalpha-, Bbeta- and gamma-chains of fibrinogen. FII(a) is a metalloproteinase, and Zn2+, Ca2+, and disulfide bonds are necessary for its fibrinogenolytic activity.
Liang
Xiu-Xia
XX
Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou 510080, China.
Zhou
Ying-Na
YN
Chen
Jia-Shu
JS
Qiu
Peng-Xin
PX
Chen
Hui-Zhen
HZ
Sun
Huan-Huan
HH
Wu
Yu-Ping
YP
Yan
Guang-Mei
GM
D017836:Agkistrodon; D000818:Animals; D002118:Calcium / Q000032:analysis; D002122:Calcium Chloride / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects*; D005343:Fibrinolytic Agents / Q000302:isolation & purification / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D008666:Metalloendopeptidases; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D011188:Potassium / Q000032:analysis; D015032:Zinc / Q000032:analysis; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D017967:Zinc Compounds; D015636:Magnesium Chloride; D004533:Egtazic Acid; C016837:zinc chloride; D005340:Fibrinogen; D004492:Edetic Acid; D045726:Metalloproteases; C478420:FIIa; D008666:Metalloendopeptidases; D015032:Zinc; D003545:Cysteine; D002122:Calcium Chloride; D011188:Potassium; D002118:Calcium; D004229:Dithiothreitol