OLIVEIRA, A. S de. Purificação e caracterização de inibidores de tripsina de sementes de Pithecellobium dumosun e seus efeitos. 222 f. 2007. Tese (Doutorado em Bioquímica)- Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2007. / Submitted by Aline Nascimento (vieiraaline@yahoo.com.br) on 2013-05-20T17:10:50Z No. of bitstreams: 1 2007_tes_asdeoliveira.pdf: 3337963 bytes, checksum: d6b49c9e4081b7dc7967d8be3fb8d65b (MD5) / Rejected by Aline Nascimento(vieiraaline@yahoo.com.br), reason: descrição on 2013-05-20T19:18:25Z (GMT) / Submitted by Aline Nascimento (vieiraaline@yahoo.com.br) on 2013-05-20T19:18:50Z No. of bitstreams: 1 2007_tes_asdeoliveira.pdf: 3337963 bytes, checksum: d6b49c9e4081b7dc7967d8be3fb8d65b (MD5) / Approved for entry into archive by Aline Nascimento(vieiraaline@yahoo.com.br) on 2013-05-20T19:19:03Z (GMT) No. of bitstreams: 1 2007_tes_asdeoliveira.pdf: 3337963 bytes, checksum: d6b49c9e4081b7dc7967d8be3fb8d65b (MD5) / Made available in DSpace on 2013-05-20T19:19:03Z (GMT). No. of bitstreams: 1 2007_tes_asdeoliveira.pdf: 3337963 bytes, checksum: d6b49c9e4081b7dc7967d8be3fb8d65b (MD5) Previous issue date: 2007 / Five Kunitz-type trypsin inhibitors (JB1, JB2, JB3-1, JB3-2 and JB4) were purified from Pithecellobium dumosum seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose and reverse phase HPLC using Vydac C-18 column. The five inhibitors had Mr between 18 and 20 kDa with a single polypeptide chain as determined by SDS-PAGE with and without reduction. JB1, JB3-1 and JB3-2 had Mr of 19.70, 19.69 and 19.69 kDa, respectively, by MALDI-TOF. JB2 and JB4 had Mr of 18.08 and 20.85, respectively, by SDS-PAGE. The N-terminal sequences of JB1, JB3-1 and JB3-2 showed identity with others Kunitz-type inhibitors. The five inhibitors were stable over a wide range of temperature and pH. The inhibition of trypsin by JB1, JB2 and JB4 was competitive. JB1, JB2, JB3-1 and JB3-2 showed Ki values of 3.56 x 10-8 M, 1.65 x 10-8 M, 4.20 x 10-8 M, 2.88 x 10-8 M, respectively, against bovine trypsin. In comparison with others inhibitors JB4, with Ki of 5.70 x 10-10 M, showed a high affinity toward trypsin. Among the inhibitors purified only JB4 inhibited chymotrypsin activity. The activities of elastase and bromelain were not inhibited for these inhibitors. The inhibition of JB1, JB2, JB3-1 and JB3-2 on papain varied between 32.93 to 48.82% of inhibition and was indicative of its bifunctionality with exception of JB4 that inhibited this activity in 9.9%. The papain inhibition by JB1 and JB2 were noncompetitive type and the Kivalues were 7.6 x 10-7 and 5.1 x 10-7 M, respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. These inhibitors were effective towards trypsin-like digestive enzymes of the insect in different degrees. The digestive enzymes from Zabrotes subfasciatus and Ceratitis capitata were inhibited by JB1 in 68.87 and 65.53% respectively, and Callosobruchus maculatus, Alabama argillaceae and Plodia intepunctella enzymes were inhibited in the range of 29.18 to 44.35%. Digestive enzymes from Z. subfasciatus, C. maculatus and C. capitata were inhibited by JB2 in the range of 70.04 to 74.54%, and the enzymes of A. argillaceae and P. intepunctella were suppressed in 13.58 and 48.67%, respectively. Trypsin-like activities of larval from Z. subfasciatus were suppressed in 67.33 and 56.93% by JB3-1 and JB3-2, respectively, and the activities of C. maculatus, A. argillaceae, P. intepunctella and C. capitata were inhibited by these inhibitors in the range of 5.17-49.00%. JB4 inhibited around 54.53 to 66.15% the digestive enzymes of C. maculatus, Z. subfasciatus and A. argillaceae and the digestive enzymes from P. intepunctella and C. capitata larvae in 8.97% and 37.47%, respectively. The inhibition of trypsin-like and papain-like proteinases of several insects suggested that these inhibitors may affect the growth and survival of these insect pests when incorporated into artificial diet and their bifunctionality are indicative that these inhibitors could be strong candidates to plant management programs cross transgenia. / Cinco inibidores de tripsina da família Kunitz (JB1, JB2, JB3-1, JB3-2 e JB4) foram purificados de sementes de Pithecellobium dumosum, uma árvore da subfamília Mimosoideae, por precipitação com ácido tricloroácetico (TCA), cromatografia de afinidade sobre tripsina imobilizada em Sepharose e coluna de fase reversa em sistema de CLAE. Os cinco inibidores possuem massa molecular entre 18 e 20 kDa formados por uma cadeia polipeptídica como determinado por SDS-PAGE na presença ou ausência de -mercaptoetanol. JB1, JB3-1 e JB3-2 têm massas moleculares de 19,70, 19,69 e 19,69 kDa, respectivamente, por MALDI-TOF. JB2 e JB4 têm massa molecular de 18,08 e 20,85 kDa, respectivamente. A seqüência N-terminal de JB1, JB3-1 e JB3-2 mostrou identidade com outros inibidores da família Kunitz. Os cinco inibidores foram estáveis às variações de temperatura e pH. A inibição da tripsina por JB1, JB2 e JB4 foi do tipo competitivo. JB1, JB2, JB3-1 e JB3-2 tiveram Kis de 3,56 x 10-8 M, 1,65 x 10-8 M, 4,20 x10-8 M, 2,88 x 10-8 M, respectivamente para a tripsina bovina. Em comparação com os outros inibidores JB4, com Ki de 5,70 x 10-10 M, apresentou maior afinidade para tripsina. Entre os inibidores purificados apenas JB4 inibiu moderadamente a atividade da quimotripsina. A atividade da elastase e bromelaína não foi inibida efetivamente por esses inibidores. A inibição de JB1, JB2, JB3-1 e JB3-2 sobre a papaína variaram entre 32,93 a 48,82% e foi indicativo de sua bifuncionalidade, com exceção de JB4 que inibiu fracamente essa atividade (9,93% de inibição). A inibição da papaína por JB1 e JB2 foi do tipo não competitiva e os valores de Ki foram de 7,6 x 10-7 e 5,1 x 10-7 M, respectivamente. Ensaios in vitro sobre as proteinases digestórias de Lepidoptera, Diptera e Coleoptera foram feitos. Esses inibidores foram efetivos para enzimas digestórias semelhantes à tripsina desses insetos em diferentes graus. As enzimas digestivas de Zabrotes subfasciatus e Ceratitis capitata foram inibidas por JB1 em 68,87 e 65,53%, respectivamente, e as enzimas de Callosobruchus maculatus, Alabama argillaceae e Plodia interpunctella foram inibidas entre 29,18 e 44,35%. Enzimas digestórias de Z. subfasciatus, C. maculatus e C. capitata foram inibidas por JB2 entre 70,04 e 74,54% e as enzimas de larvas de A argillaceae e P. interpunctella foram suprimidas em 13,58 e 48,67%, respectivamente. A atividade semelhante à tripsina de larvas de Z. subfasciatus foi suprimida em 67,33 e 56,93% por JB3-1 e JB3-2, respectivamente, e a atividade de C. maculatus, A argillaceae, P. interpunctella e C. capitata foram suprimidas por esses inibidores entre 5,17 e 49,00%. JB4 inibiu entre 54,53 a 66,15 % as enzimas digestivas de C. maculatus, Z. subfasciatus e A argillaceae e inibiu as enzimas digestivas de larvas de P. intepunctella e C. capitata em 8,97 e 37,47%, respectivamente. A inibição de proteinases semelhantes à tripsina e à papaína presentes no intestino de vários insetos sugere que esses inibidores possam afetar o crescimento e sobrevivência desses insetos pragas quando incorporados em sementes artificiais e esta bifuncionalidade é indicativo de que estes inibidores possam ser fortes candidatos para os programas de melhoramento de plantas via transgenia.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Cinco inibidores de tripsina da famÃlia Kunitz (JB1, JB2, JB3-1, JB3-2 e JB4) foram purificados de sementes de Pithecellobium dumosum, uma Ãrvore da subfamÃlia Mimosoideae, por precipitaÃÃo com Ãcido tricloroÃcetico (TCA), cromatografia de afinidade sobre tripsina imobilizada em Sepharose e coluna de fase reversa em sistema de CLAE. Os cinco inibidores possuem massa molecular entre 18 e 20 kDa formados por uma cadeia polipeptÃdica como determinado por SDS-PAGE na presenÃa ou ausÃncia de -mercaptoetanol. JB1, JB3-1 e JB3-2 tÃm massas moleculares de 19,70, 19,69 e 19,69 kDa, respectivamente, por MALDI-TOF. JB2 e JB4 tÃm massa molecular de 18,08 e 20,85 kDa, respectivamente. A seqÃÃncia N-terminal de JB1, JB3-1 e JB3-2 mostrou identidade com outros inibidores da famÃlia Kunitz. Os cinco inibidores foram estÃveis Ãs variaÃÃes de temperatura e pH. A inibiÃÃo da tripsina por JB1, JB2 e JB4 foi do tipo competitivo. JB1, JB2, JB3-1 e JB3-2 tiveram Kis de 3,56 x 10-8 M, 1,65 x 10-8 M, 4,20 x10-8 M, 2,88 x 10-8 M, respectivamente para a tripsina bovina. Em comparaÃÃo com os outros inibidores JB4, com Ki de 5,70 x 10-10 M, apresentou maior afinidade para tripsina. Entre os inibidores purificados apenas JB4 inibiu moderadamente a atividade da quimotripsina. A atividade da elastase e bromelaÃna nÃo foi inibida efetivamente por esses inibidores. A inibiÃÃo de JB1, JB2, JB3-1 e JB3-2 sobre a papaÃna variaram entre 32,93 a 48,82% e foi indicativo de sua bifuncionalidade, com exceÃÃo de JB4 que inibiu fracamente essa atividade (9,93% de inibiÃÃo). A inibiÃÃo da papaÃna por JB1 e JB2 foi do tipo nÃo competitiva e os valores de Ki foram de 7,6 x 10-7 e 5,1 x 10-7 M, respectivamente. Ensaios in vitro sobre as proteinases digestÃrias de Lepidoptera, Diptera e Coleoptera foram feitos. Esses inibidores foram efetivos para enzimas digestÃrias semelhantes à tripsina desses insetos em diferentes graus. As enzimas digestivas de Zabrotes subfasciatus e Ceratitis capitata foram inibidas por JB1 em 68,87 e 65,53%, respectivamente, e as enzimas de Callosobruchus maculatus, Alabama argillaceae e Plodia interpunctella foram inibidas entre 29,18 e 44,35%. Enzimas digestÃrias de Z. subfasciatus, C. maculatus e C. capitata foram inibidas por JB2 entre 70,04 e 74,54% e as enzimas de larvas de A argillaceae e P. interpunctella foram suprimidas em 13,58 e 48,67%, respectivamente. A atividade semelhante à tripsina de larvas de Z. subfasciatus foi suprimida em 67,33 e 56,93% por JB3-1 e JB3-2, respectivamente, e a atividade de C. maculatus, A argillaceae, P. interpunctella e C. capitata foram suprimidas por esses inibidores entre 5,17 e 49,00%. JB4 inibiu entre 54,53 a 66,15 % as enzimas digestivas de C. maculatus, Z. subfasciatus e A argillaceae e inibiu as enzimas digestivas de larvas de P. intepunctella e C. capitata em 8,97 e 37,47%, respectivamente. A inibiÃÃo de proteinases semelhantes à tripsina e à papaÃna presentes no intestino de vÃrios insetos sugere que esses inibidores possam afetar o crescimento e sobrevivÃncia desses insetos pragas quando incorporados em sementes artificiais e esta bifuncionalidade à indicativo de que estes inibidores possam ser fortes candidatos para os programas de melhoramento de plantas via transgenia. / Five Kunitz-type trypsin inhibitors (JB1, JB2, JB3-1, JB3-2 and JB4) were purified from Pithecellobium dumosum seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose and reverse phase HPLC using Vydac C-18 column. The five inhibitors had Mr between 18 and 20 kDa with a single polypeptide chain as determined by SDS-PAGE with and without reduction. JB1, JB3-1 and JB3-2 had Mr of 19.70, 19.69 and 19.69 kDa, respectively, by MALDI-TOF. JB2 and JB4 had Mr of 18.08 and 20.85, respectively, by SDS-PAGE. The N-terminal sequences of JB1, JB3-1 and JB3-2 showed identity with others Kunitz-type inhibitors. The five inhibitors were stable over a wide range of temperature and pH. The inhibition of trypsin by JB1, JB2 and JB4 was competitive. JB1, JB2, JB3-1 and JB3-2 showed Ki values of 3.56 x 10-8 M, 1.65 x 10-8 M, 4.20 x 10-8 M, 2.88 x 10-8 M, respectively, against bovine trypsin. In comparison with others inhibitors JB4, with Ki of 5.70 x 10-10 M, showed a high affinity toward trypsin. Among the inhibitors purified only JB4 inhibited chymotrypsin activity. The activities of elastase and bromelain were not inhibited for these inhibitors. The inhibition of JB1, JB2, JB3-1 and JB3-2 on papain varied between 32.93 to 48.82% of inhibition and was indicative of its bifunctionality with exception of JB4 that inhibited this activity in 9.9%. The papain inhibition by JB1 and JB2 were noncompetitive type and the Kivalues were 7.6 x 10-7 and 5.1 x 10-7 M, respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. These inhibitors were effective towards trypsin-like digestive enzymes of the insect in different degrees. The digestive enzymes from Zabrotes subfasciatus and Ceratitis capitata were inhibited by JB1 in 68.87 and 65.53% respectively, and Callosobruchus maculatus, Alabama argillaceae and Plodia intepunctella enzymes were inhibited in the range of 29.18 to 44.35%. Digestive enzymes from Z. subfasciatus, C. maculatus and C. capitata were inhibited by JB2 in the range of 70.04 to 74.54%, and the enzymes of A. argillaceae and P. intepunctella were suppressed in 13.58 and 48.67%, respectively. Trypsin-like activities of larval from Z. subfasciatus were suppressed in 67.33 and 56.93% by JB3-1 and JB3-2, respectively, and the activities of C. maculatus, A. argillaceae, P. intepunctella and C. capitata were inhibited by these inhibitors in the range of 5.17-49.00%. JB4 inhibited around 54.53 to 66.15% the digestive enzymes of C. maculatus, Z. subfasciatus and A. argillaceae and the digestive enzymes from P. intepunctella and C. capitata larvae in 8.97% and 37.47%, respectively. The inhibition of trypsin-like and papain-like proteinases of several insects suggested that these inhibitors may affect the growth and survival of these insect pests when incorporated into artificial diet and their bifunctionality are indicative that these inhibitors could be strong candidates to plant management programs cross transgenia.