RESUMO
A novel fibrinogenolytic protease, named alpha-mucrofibrase, was purified from the venom of Chinese Habu (Trimeresurus mucrosquamatus) by DEAE-Sephadex A-50 ion-exchange chromatography and Sephadex G-100 (super fine) gel filtration alpha-Mucrofibrase is a single-chain polypeptide of approximately 29 kDa. It is stable even at 95 degrees C, and the most susceptible hydrolysis substrate is S-2302. It cleaved primarily the Aalpha chain of fibrinogen followed by the Bbeta chain, while the gamma chain was partially affected. N-terminal sequence of this fibrinogenolytic enzyme has great homology with those of other snake venom serine proteases. The esterase activity of alpha-mucrofibrase is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by metal chelator (EDTA), suggesting this fibrinogenase belongs to the venom serine protease family.
Assuntos
Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/isolamento & purificação , Metaloendopeptidases/isolamento & purificação , Serina Endopeptidases/isolamento & purificação , Trimeresurus , Sequência de Aminoácidos , Animais , Cromatografia DEAE-Celulose , Cromatografia por Troca Iônica , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Estabilidade Enzimática , Fibrinogênio/metabolismo , Hemorragia/induzido quimicamente , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Camundongos , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Coelhos , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Especificidade por SubstratoRESUMO
Several biochemical and biological activities such as phospholipase A2, arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA2) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA2 had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA2 was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.