PURIFICATION, BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF THE THROMBIN-LIKE ENZYME PRESENT IN THE VENOM OFTHE SNAKE Bothrops brazili

• Autores: Ruiz, Luis, Vivas Ruiz, Dan, Lazo, Fanny, Seifert, Wolfram, Rodríguez, Edith, Sandoval, Gustavo, Yarlequé, Armando
• Formato: artículo
• Fecha de Publicación: 2017
• Institución: Sociedad Química del Perú
• Repositorio: Revista de la Sociedad Química del Perú
• Lenguaje: español
• OAI Identifier: oai:rsqp.revistas.sqperu.org.pe:article/218
• Enlace del recurso: http://revistas.sqperu.org.pe/index.php/revistasqperu/article/view/218
• Nivel de acceso: acceso abierto
• Materia: Bothrops brazili; Thrombin-like enzyme; snake; venom; coagulation; enzima similar a trombina; veneno; serpiente; coagulación

A coagulant enzyme from Bothrops brazili snake venom called thrombin-like enzyme was purified by three successive chromatographic steps on Sephadex G-75, DEAE Sephadex A-50 and Sephadex G-50 using 0.05M Tris-HCl buffer pH 8.5. The enzyme was purified 15.9 times with a yield of 28.6% and by PAGE-SDS a single protein band of 48 kDa was obtained both in reducing and non-reducing conditions using 2β-Mercaptoethano., It is a unicatenaryprotein with coagulant activity on both citrated human plasma and bovine fibrinogen. The enzyme showed amidolytic activity on the chromogenic substrate Benzoyl-Arginyl-pNitroaniline (BApNA) and the coagulant potency on bovine fibrinogen was calculated on 121 NIH units of thrombin / mg. The enzyme was inhibited by PMSF and the soybean trypsin inhibitor, therefore, it is a serine protease; the optimum pH for the amidolytic activity was 8.5 and the protein was stable to heat treatment only up to 40 °C. The minimal defibrinogenating dose was 8 μg / g of mouse and by double immunodiffusion tests immunoreactivity was observed with respect to INS polyvalent antibothropic serum.