Utilização do inibidor de papaína extraído de sementes de Adenanthera pavonina L. na purificação de proteases cisteínicas

• Autor(a) principal: GAMBÔA, Adriane Guimarães
• Data de Publicação: 2010
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• dARK ID: ark:/38995/001300000b1kp
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tde/1238
• topic: A. pavonina; polianilina; proteases cisteínicas; A. pavonina; polyaniline; cysteine proteases; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

In this work papain inhibitor from A. pavonina was immobilized by covalent bond in polyaniline (PANI) modified with glutaraldehyde (PANIG) for be used as stationary phase for affinity chromatography and then applied in the purification of cysteine proteases bromelain and ficin. The extraction and purification of inhibitors protease from A. pavonina resulted in a yield of 3.9% in the last step of purification. Gel filtration chromatography performed in Sephadex G-75 resin as a purification step resulted in three protein peaks (F1, F2 and F3), but only F1 was used in the experiments of immobilization because of higher specific activity. Immobilization was performed using PANIG. To optimize the immobilization conditions the amount of PANIG in the reaction (5, 10 and 15mg), time (30, 60 and 90 min) and pH (5.0 to 8.0) were varied. The best conditions for immobilization of A. pavonina inhibitor, according to tests performed were 5mg PANIG, reaction time of 30min and pH 7.0. PANIG-I was used as bio-affinity stationary phase for separation of bromelain and ficin. Electrophoresis (SDS-PAGE) performed after the separation process revealed the presence of a single band for both bromelain and ficina, with 28 and 25 kDa, respectively. In this process, ficin was purified 2,60 fold and bromelain 0,89 fold, showing that the use of inhibitors of A. pavonina immobilized in PANIG were efficient in the purification of cysteine proteases.