Purification, characterization and crystallization of Jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu.
A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoelectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIHU/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degrees C and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 degrees C. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4A.
Bortoleto
Raquel K
RK
Department of Physics, IBILCE/UNESP, Cristovão Colombo, 2265, Caixa Postal 136, SP, São José do Rio Preto 15054-000, Brazil.
Murakami
Mário T
MT
Watanabe
Leandra
L
Soares
Andreimar M
AM
Arni
Raghuvir K
RK
D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D017837:Bothrops; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D001550:Benzamidines; D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D011480:Protease Inhibitors; D005340:Fibrinogen; D004950:Esterases; D012697:Serine Endopeptidases; C032157:benzamidine; D004229:Dithiothreitol