Purifica??o, caracteriza??o e atividade bioinseticida de um inibidor de tripsina de sementes de Crotalaria pallida

• Main Author: Gomes, Carlos Eduardo Maia
• Other Authors: CPF:29706106391
• Format: Others
• Language: Portuguese
• Published: Universidade Federal do Rio Grande do Norte 2014
• Subjects: Prote?nas - bioqu?mica; Inibidores de tripsina - sementes; Atividade biol?gica - enzimas digestivas; Cultura agr?cola - nordeste; Bioss?ntese; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
• Online Access: http://repositorio.ufrn.br:8080/jspui/handle/123456789/12530

Made available in DSpace on 2014-12-17T14:03:27Z (GMT). No. of bitstreams: 1 CarlosEMG.pdf: 434119 bytes, checksum: cffd1dddf77ec049b23dadc1bfa89773 (MD5) Previous issue date: 2004-12-20 === Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior === A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulphate fractionation, affinity chromatography on immobilized Trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named ITC, had Mr of 32.5 kDa by SDS-PAGE and was composed by two subunits with 27.7 and 5.6 kDa linked by disulphide bridges, a typical characteristic of Kunitz-Inhibitor family. ITC was stable until 50?C, and at 100?C its residual activity was of about 60%. Also, ITC was stable at pHs 2 to 12. The inhibition of trypsin by ITC was non-competitive, with a Ki of 8,8 x 10-7M. ITC inhibits weakly other serine proteinases such as chymotrypsin and elastase. The inhibition of papain (44% of inhibition), a cysteine proteinase was an indicative of the bi-functionality of ITC. In vitro assays against digestive proteinases from several Lepdoptera, Diptera and Coleoptera pests were made. ITC inhibited in 100% digestive enzymes of Ceratitis capitata (fruit fly), Spodoptera frugiperda and Alabama argillacea, the last one being a cotton pest. It also inhibited in 74.4% Callosobruchus maculatus (bean weevil) digestive enzymes, a Coleoptera pest. ITC, when added in artificial diet models, affected weakly the development of C. capitata larvae and it had a WD50 of 2.65% to C. maculatus larvae === Um inibidor de proteinase foi isolado de sementes de Crotalaria pallida por fracionamento com sulfato de am?nio, cromatografia de afinidade em Sepharose com tripsina acoplada e precipita??o com ?cido tricloroac?tico (TCA). O inibidor de tripsina, denominado ITC, possui massa molecular de 32,5 kDa determinada por SDS-PAGE e ? composto por duas subunidades, uma de 25,7 e outra de 5,6 kDa, ligadas por pontes dissulfeto; uma caracter?stica t?pica da fam?lia de inibidores de Kunitz. ITC foi est?vel at? 50?C e a 100?C sua atividade residual foi cerca de 60%. ITC tamb?m foi est?vel em valores de pHs variando de 2 a 12. A inibi??o da tripsina por ITC ? do tipo n?o-competitivo, com um Ki de 8,8 x 10-7M. ITC inibe fracamente outras proteinases ser?nicas como quimotripsina e elastase. A inibi??o da papa?na (44%), uma proteinase ciste?nica, foi indicativa da bifuncionalidade de ITC. Ensaios in vitro contra proteinases digestivas de v?rios insetos praga das ordens Lepidoptera, Diptera e Coleoptera foram realizados. ITC inibiu em cerca de 100% enzimas digestivas de Ceratitis capitata (mosca-das-frutas), Spodoptera frugiperda (lagarta do cartucho do algod?o), e Alabama argillacea (curuquer? do algod?o). ITC tamb?m inibiu em 74,4% enzimas digestivas de Callosobruchus maculatus (broca do feij?o) uma praga da ordem Coleoptera. ITC, quando adicionado a dietas artificiais, afetou fracamente o desenvolvimento de larvas de C. capitata e teve um WD50 de 2,65% contra larvas de C. maculatus