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Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom |
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| Authors: | Júnia de O Costa Kelly C Fonseca Marcelo E Beletti Andreimar M Soares Silvia NS Hirayama Fernando Fonseca Nilson Penha-Silva |
| Institution: | a Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, 38400-902 Uberlândia-MG, Brazil b Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, 38400-902 Uberlândia-MG, Brazil c Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, FMRP-USP, Ribeirão Preto-SP, Brazil d Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto-SP, Brazil e Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto-SP, Brazil f Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, SP, Brazil g Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brazil h Instituto Federal de Educação, Ciência e Tecnologia do Triângulo Mineiro, Campus Ituiutaba, Ituiutaba-MG, Brazil i Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica (N-Biofar), Brazil |
| Abstract: | A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708 bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 °C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aα-chain, apparently not degrading the Bβ and γ-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 °C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders. |
| Keywords: | Bothrops alternatus Snake venom Fibrinogenase Thrombin-like Defibrinogenation |
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