Purification from Bothrops lanceolatus (fer de lance) venom of a fibrino(geno)lytic enzyme with esterolytic activity.
Bothrops lanceolatus venom has high caseinolytic, phospholipasic, esterolytic and hemorrhagic activities. In spite of having no coagulant effect on plasma, this venom contains a thrombin-like enzyme. Using gel filtration and ion-exchange chromatographies, we have purified an esterolytic fraction (F-II-1a) from this venom with a protein yield of 4% and a 58% recovery in enzyme activity. SDS-PAGE in the presence of beta-mercaptoethanol showed that the enzyme is a single chain polypeptide with a MW=38,100. Immunodiffusion and immunoelectrophoresis of fraction F-II-1a against serum from horses immunized with B. lanceolatus venom and against rabbit antiserum prepared using fraction F-II-1a both showed a single immunoprecipitin line. The Km and Vmax values for TAME hydrolysis were 0.85 mM and 38.6 micromol/min/mg, respectively. The esterolytic activity was completely inhibited by PMSF (10 mM) but not by EDTA (20 mM). Fraction F-II-1a hydrolyzed the alpha and beta chains of fibrinogen. Degradation of the alpha chain occurred within 10 min while that of the beta-chain was slower. The enzyme had no effect on the gamma-chain even after 4 h of hydrolysis.
Lôbo de Araújo
A
A
Departamento de Farmacologia, FCM, CP 6111, UNICAMP, Campinas, SP, Brasil.
Donato
J L
JL
Bon
C
C
D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000201:enzymology*; D004950:Esterases / Q000302:isolation & purification* / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D008970:Molecular Weight; D011817:Rabbits; D013917:Thrombin / Q000378:metabolism; D014106:Tosylarginine Methyl Ester / Q000378:metabolism
D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D014106:Tosylarginine Methyl Ester; D004950:Esterases; D013917:Thrombin