Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong.
Autor(a) principal: | |
---|---|
Data de Publicação: | 2014 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da UFC |
Texto Completo: | http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=15542 |
Resumo: | Protease inhibitors are proteins with the intrinsic ability to inhibit catalytic activity of enzymes and are very common in plant seeds. Proteases play a major role in development of several diseases. The control of their activity by protease inhibitors have increased interest on these molecules as chemopreventive agents, specially for cancer. The anticarcinogenic effects of legume seeds present in human diet as well as those of underexploited plant species have been extensively investigated. This study aimed to purify, perform biochemical characterization and evaluate the in vitro chemopreventive potential of protease inhibitors from Enterolobium contortisiliquum seeds upon human colorectal adenocarcinoma cells. Two protease inhibitors were purified and named EcCI and EcTI. By means of N-terminal sequence analysis, EcCI was identified as a Kunitz type inhibitor. EcTI was identified, by means of peptide mass fingerprinting and N-terminal sequence analyses, as the same EcTI purified previously (NCBI Protein BLAST; access number: sp|P86451.1|ITRY_ENTCO). The molecular masses of the two inhibitors determined by means of SDS-PAGE and mass spectrometry are, respectively, 18.5 kDa and 19,710.4 Da (EcCI); 20.2 kDa and 19,813.22 Da (EcTI). Both inhibitors consist of two polypeptide chains and have several isoforms with acidic pIs, ranging from 5 to 6. Towards chymotrypsin, EcCI is a non competitive inhibitor and shows ki of 8 x 10-8 M, while EcTI is a competitive inhibitor with ki of 48 x 10-8 M. Towards trypsin, EcTI shows non competitive inhibition and ki of 2.8 x 10-8 M. EcCI and EcTI show high (93%) and moderate (38%) chymotrypsin inhibition and EcTI was also able to strongly inhibit trypsin (100%). EcCI and EcTI showed high leukocyte elastase inhibition (85 and 75%), respectively, and inhibited pancreatic elastase weakly (about 10%). Neither of them was able to inhibit papain nor bromelain. Both inhibitors are functionally stable under wide temperature range (from 37 to 70 ÂC â EcCI; from 37 to 60 ÂC â EcTI), pH (from 2 to 12) and DTT concentration (from 1 to 10 mM â EcCI; from 1 to 100 mM â EcTI). Both EcCI and EcTI were able to inhibit HT29 colorectal adenocarcinoma cells with IC50 of 35.5 and 20.4 x 10-6 M, respectively. These results clearly indicate that these are molecules with interesting biotechnological features and very promising tools as chemopreventive agents. |
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Biblioteca Digital de Teses e Dissertações da UFC |
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info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisPurification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong.PurificaÃÃo, caracterizaÃÃo bioquÃmica e potencial quimiopreventivo de um novo inibidor de quimotripsina de sementes de Enterolobium contortisiliquum (Vell.) Morong.2014-09-18Ana de FÃtima Fontenele Urano Carvalho11804270334http://lattes.cnpq.br/2882990621973152Alfonso ClementeAAF258603NÃo informadoIlka Maria Vasconcelos31109128304http://lattes.cnpq.br/0103197383336584Ana Cristina de Oliveira Monteiro Moreira42420199391http://lattes.cnpq.br/3183895586263436Frederico Bruno Mendes Batista Moreno76121380349http://lattes.cnpq.br/858649480744672662335073320http://lattes.cnpq.br/9691278485378801Lady Clarissa Brito da Rocha BezerraUniversidade Federal do CearÃPrograma de PÃs-GraduaÃÃo em BioquÃmicaUFCBRCÃncer TimbaÃba Inibidor de proteaseCancer TimbaÃba Protease inhibitorBIOQUIMICAProtease inhibitors are proteins with the intrinsic ability to inhibit catalytic activity of enzymes and are very common in plant seeds. Proteases play a major role in development of several diseases. The control of their activity by protease inhibitors have increased interest on these molecules as chemopreventive agents, specially for cancer. The anticarcinogenic effects of legume seeds present in human diet as well as those of underexploited plant species have been extensively investigated. This study aimed to purify, perform biochemical characterization and evaluate the in vitro chemopreventive potential of protease inhibitors from Enterolobium contortisiliquum seeds upon human colorectal adenocarcinoma cells. Two protease inhibitors were purified and named EcCI and EcTI. By means of N-terminal sequence analysis, EcCI was identified as a Kunitz type inhibitor. EcTI was identified, by means of peptide mass fingerprinting and N-terminal sequence analyses, as the same EcTI purified previously (NCBI Protein BLAST; access number: sp|P86451.1|ITRY_ENTCO). The molecular masses of the two inhibitors determined by means of SDS-PAGE and mass spectrometry are, respectively, 18.5 kDa and 19,710.4 Da (EcCI); 20.2 kDa and 19,813.22 Da (EcTI). Both inhibitors consist of two polypeptide chains and have several isoforms with acidic pIs, ranging from 5 to 6. Towards chymotrypsin, EcCI is a non competitive inhibitor and shows ki of 8 x 10-8 M, while EcTI is a competitive inhibitor with ki of 48 x 10-8 M. Towards trypsin, EcTI shows non competitive inhibition and ki of 2.8 x 10-8 M. EcCI and EcTI show high (93%) and moderate (38%) chymotrypsin inhibition and EcTI was also able to strongly inhibit trypsin (100%). EcCI and EcTI showed high leukocyte elastase inhibition (85 and 75%), respectively, and inhibited pancreatic elastase weakly (about 10%). Neither of them was able to inhibit papain nor bromelain. Both inhibitors are functionally stable under wide temperature range (from 37 to 70 ÂC â EcCI; from 37 to 60 ÂC â EcTI), pH (from 2 to 12) and DTT concentration (from 1 to 10 mM â EcCI; from 1 to 100 mM â EcTI). Both EcCI and EcTI were able to inhibit HT29 colorectal adenocarcinoma cells with IC50 of 35.5 and 20.4 x 10-6 M, respectively. These results clearly indicate that these are molecules with interesting biotechnological features and very promising tools as chemopreventive agents.Inibidores de proteases sÃo proteÃnas que inibem a atividade catalÃtica de enzimas, sendo bastante comuns em sementes de plantas. As proteases desempenham papÃis centrais no desenvolvimento de muitas doenÃas. O controle de sua atividade realizado por inibidores de proteases despertou o interesse sobre estas molÃculas como agentes quimiopreventivos, especialmente sobre o cÃncer. Os efeitos anticarcinogÃnicos das sementes de leguminosas presentes comumente na dieta, bem como de espÃcies vegetais subexploradas, tÃm sido extensivamente investigados. O objetivo deste trabalho foi purificar, caracterizar bioquimicamente e avaliar o potencial quimiopreventivo in vitro de inibidores de proteases de sementes de Enterolobium contortisiliquum, utilizando como modelo cÃlulas de adenocarcinoma colorretal humano. Foram purificados dois inibidores de proteases denominados EcCI e EcTI. AtravÃs da sequÃncia N-terminal, EcCI foi identificado como um inibidor da famÃlia Kunitz. EcTI foi identificado, por meio de peptide mass fingerprinting e por anÃlise da sequÃncia N-terminal, como aquele descrito previamente na literatura (NCBI Protein BLAST; nÃmero de acesso: sp|P86451.1|ITRY_ENTCO). Suas massas moleculares determinadas por SDS-PAGE e espectrometria de massas sÃo, respectivamente, 18,5 kDa e 19.710,4 Da (EcCI); 20,2 kDa e 19.813,22 Da (EcTI). Ambos os inibidores sÃo formados de duas subunidades proteicas e apresentam isoformas cujos pIâs sÃo Ãcidos, entre 5 e 6. Frente a quimotripsina, EcCI à um inibidor nÃo competitivo e apresenta ki de 8 x 10-8 M, enquanto EcTI à inibidor competitivo com ki de 48 x 10-8 M. Frente à tripsina, EcTI apresenta inibiÃÃo nÃo competitiva e ki de 2,8 x 10-8 M. EcCI e EcTI apresentam atividade inibitÃria de quimotripsina alta (93%) e moderada (38%), respectivamente. Apenas EcTI foi capaz de inibir a tripsina (100%). EcCI e EcTI apresentam alta atividade inibitÃria de elastase neutrofÃlica (85 e 75%, respectivamente). Os dois inibidores inibiram sutilmente a elastase pancreÃtica (ca. de 10%) e nenhum foi capaz de inibir papaÃna e bromelaÃna. Os dois inibidores apresentam alta estabilidade à variaÃÃo de temperatura (de 37 a 70 ÂC para EcCI e de 37 a 60 ÂC para EcTI), pH (2 a 12) e concentraÃÃo de DTT (de 1 a 10 mM para EcCI e de 1 a 100 mM para EcTI). EcCI e EcTI inibiram a proliferaÃÃo de cÃlulas de adenocarcinoma colorretal humano da linhagem HT29 com CI50 de 35,5 e 20,4 x 10-6 M, respectivamente, indicando que esses inibidores apresentam bom potencial quimiopreventivo e, portanto, sÃo molÃculas bastante interessantes do ponto de vista biotecnolÃgico. CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superiorhttp://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=15542application/pdfinfo:eu-repo/semantics/openAccessporreponame:Biblioteca Digital de Teses e Dissertações da UFCinstname:Universidade Federal do Cearáinstacron:UFC2019-01-21T11:28:46Zmail@mail.com - |
dc.title.en.fl_str_mv |
Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong. |
dc.title.alternative.pt.fl_str_mv |
PurificaÃÃo, caracterizaÃÃo bioquÃmica e potencial quimiopreventivo de um novo inibidor de quimotripsina de sementes de Enterolobium contortisiliquum (Vell.) Morong. |
title |
Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong. |
spellingShingle |
Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong. Lady Clarissa Brito da Rocha Bezerra CÃncer TimbaÃba Inibidor de protease Cancer TimbaÃba Protease inhibitor BIOQUIMICA |
title_short |
Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong. |
title_full |
Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong. |
title_fullStr |
Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong. |
title_full_unstemmed |
Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong. |
title_sort |
Purification, Biochemical Characterization and chemopreventive potential hum new inhibitor of chymotrypsin Enterolobium seeds contortisiliquum (Vell.) Morong. |
author |
Lady Clarissa Brito da Rocha Bezerra |
author_facet |
Lady Clarissa Brito da Rocha Bezerra |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Ana de FÃtima Fontenele Urano Carvalho |
dc.contributor.advisor1ID.fl_str_mv |
11804270334 |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/2882990621973152 |
dc.contributor.advisor-co1.fl_str_mv |
Alfonso Clemente |
dc.contributor.advisor-co1ID.fl_str_mv |
AAF258603 |
dc.contributor.advisor-co1Lattes.fl_str_mv |
NÃo informado |
dc.contributor.referee1.fl_str_mv |
Ilka Maria Vasconcelos |
dc.contributor.referee1ID.fl_str_mv |
31109128304 |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/0103197383336584 |
dc.contributor.referee2.fl_str_mv |
Ana Cristina de Oliveira Monteiro Moreira |
dc.contributor.referee2ID.fl_str_mv |
42420199391 |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/3183895586263436 |
dc.contributor.referee3.fl_str_mv |
Frederico Bruno Mendes Batista Moreno |
dc.contributor.referee3ID.fl_str_mv |
76121380349 |
dc.contributor.referee3Lattes.fl_str_mv |
http://lattes.cnpq.br/8586494807446726 |
dc.contributor.authorID.fl_str_mv |
62335073320 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/9691278485378801 |
dc.contributor.author.fl_str_mv |
Lady Clarissa Brito da Rocha Bezerra |
contributor_str_mv |
Ana de FÃtima Fontenele Urano Carvalho Alfonso Clemente Ilka Maria Vasconcelos Ana Cristina de Oliveira Monteiro Moreira Frederico Bruno Mendes Batista Moreno |
dc.subject.por.fl_str_mv |
CÃncer TimbaÃba Inibidor de protease |
topic |
CÃncer TimbaÃba Inibidor de protease Cancer TimbaÃba Protease inhibitor BIOQUIMICA |
dc.subject.eng.fl_str_mv |
Cancer TimbaÃba Protease inhibitor |
dc.subject.cnpq.fl_str_mv |
BIOQUIMICA |
dc.description.sponsorship.fl_txt_mv |
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior |
dc.description.abstract.por.fl_txt_mv |
Protease inhibitors are proteins with the intrinsic ability to inhibit catalytic activity of enzymes and are very common in plant seeds. Proteases play a major role in development of several diseases. The control of their activity by protease inhibitors have increased interest on these molecules as chemopreventive agents, specially for cancer. The anticarcinogenic effects of legume seeds present in human diet as well as those of underexploited plant species have been extensively investigated. This study aimed to purify, perform biochemical characterization and evaluate the in vitro chemopreventive potential of protease inhibitors from Enterolobium contortisiliquum seeds upon human colorectal adenocarcinoma cells. Two protease inhibitors were purified and named EcCI and EcTI. By means of N-terminal sequence analysis, EcCI was identified as a Kunitz type inhibitor. EcTI was identified, by means of peptide mass fingerprinting and N-terminal sequence analyses, as the same EcTI purified previously (NCBI Protein BLAST; access number: sp|P86451.1|ITRY_ENTCO). The molecular masses of the two inhibitors determined by means of SDS-PAGE and mass spectrometry are, respectively, 18.5 kDa and 19,710.4 Da (EcCI); 20.2 kDa and 19,813.22 Da (EcTI). Both inhibitors consist of two polypeptide chains and have several isoforms with acidic pIs, ranging from 5 to 6. Towards chymotrypsin, EcCI is a non competitive inhibitor and shows ki of 8 x 10-8 M, while EcTI is a competitive inhibitor with ki of 48 x 10-8 M. Towards trypsin, EcTI shows non competitive inhibition and ki of 2.8 x 10-8 M. EcCI and EcTI show high (93%) and moderate (38%) chymotrypsin inhibition and EcTI was also able to strongly inhibit trypsin (100%). EcCI and EcTI showed high leukocyte elastase inhibition (85 and 75%), respectively, and inhibited pancreatic elastase weakly (about 10%). Neither of them was able to inhibit papain nor bromelain. Both inhibitors are functionally stable under wide temperature range (from 37 to 70 ÂC â EcCI; from 37 to 60 ÂC â EcTI), pH (from 2 to 12) and DTT concentration (from 1 to 10 mM â EcCI; from 1 to 100 mM â EcTI). Both EcCI and EcTI were able to inhibit HT29 colorectal adenocarcinoma cells with IC50 of 35.5 and 20.4 x 10-6 M, respectively. These results clearly indicate that these are molecules with interesting biotechnological features and very promising tools as chemopreventive agents. Inibidores de proteases sÃo proteÃnas que inibem a atividade catalÃtica de enzimas, sendo bastante comuns em sementes de plantas. As proteases desempenham papÃis centrais no desenvolvimento de muitas doenÃas. O controle de sua atividade realizado por inibidores de proteases despertou o interesse sobre estas molÃculas como agentes quimiopreventivos, especialmente sobre o cÃncer. Os efeitos anticarcinogÃnicos das sementes de leguminosas presentes comumente na dieta, bem como de espÃcies vegetais subexploradas, tÃm sido extensivamente investigados. O objetivo deste trabalho foi purificar, caracterizar bioquimicamente e avaliar o potencial quimiopreventivo in vitro de inibidores de proteases de sementes de Enterolobium contortisiliquum, utilizando como modelo cÃlulas de adenocarcinoma colorretal humano. Foram purificados dois inibidores de proteases denominados EcCI e EcTI. AtravÃs da sequÃncia N-terminal, EcCI foi identificado como um inibidor da famÃlia Kunitz. EcTI foi identificado, por meio de peptide mass fingerprinting e por anÃlise da sequÃncia N-terminal, como aquele descrito previamente na literatura (NCBI Protein BLAST; nÃmero de acesso: sp|P86451.1|ITRY_ENTCO). Suas massas moleculares determinadas por SDS-PAGE e espectrometria de massas sÃo, respectivamente, 18,5 kDa e 19.710,4 Da (EcCI); 20,2 kDa e 19.813,22 Da (EcTI). Ambos os inibidores sÃo formados de duas subunidades proteicas e apresentam isoformas cujos pIâs sÃo Ãcidos, entre 5 e 6. Frente a quimotripsina, EcCI à um inibidor nÃo competitivo e apresenta ki de 8 x 10-8 M, enquanto EcTI à inibidor competitivo com ki de 48 x 10-8 M. Frente à tripsina, EcTI apresenta inibiÃÃo nÃo competitiva e ki de 2,8 x 10-8 M. EcCI e EcTI apresentam atividade inibitÃria de quimotripsina alta (93%) e moderada (38%), respectivamente. Apenas EcTI foi capaz de inibir a tripsina (100%). EcCI e EcTI apresentam alta atividade inibitÃria de elastase neutrofÃlica (85 e 75%, respectivamente). Os dois inibidores inibiram sutilmente a elastase pancreÃtica (ca. de 10%) e nenhum foi capaz de inibir papaÃna e bromelaÃna. Os dois inibidores apresentam alta estabilidade à variaÃÃo de temperatura (de 37 a 70 ÂC para EcCI e de 37 a 60 ÂC para EcTI), pH (2 a 12) e concentraÃÃo de DTT (de 1 a 10 mM para EcCI e de 1 a 100 mM para EcTI). EcCI e EcTI inibiram a proliferaÃÃo de cÃlulas de adenocarcinoma colorretal humano da linhagem HT29 com CI50 de 35,5 e 20,4 x 10-6 M, respectivamente, indicando que esses inibidores apresentam bom potencial quimiopreventivo e, portanto, sÃo molÃculas bastante interessantes do ponto de vista biotecnolÃgico. |
description |
Protease inhibitors are proteins with the intrinsic ability to inhibit catalytic activity of enzymes and are very common in plant seeds. Proteases play a major role in development of several diseases. The control of their activity by protease inhibitors have increased interest on these molecules as chemopreventive agents, specially for cancer. The anticarcinogenic effects of legume seeds present in human diet as well as those of underexploited plant species have been extensively investigated. This study aimed to purify, perform biochemical characterization and evaluate the in vitro chemopreventive potential of protease inhibitors from Enterolobium contortisiliquum seeds upon human colorectal adenocarcinoma cells. Two protease inhibitors were purified and named EcCI and EcTI. By means of N-terminal sequence analysis, EcCI was identified as a Kunitz type inhibitor. EcTI was identified, by means of peptide mass fingerprinting and N-terminal sequence analyses, as the same EcTI purified previously (NCBI Protein BLAST; access number: sp|P86451.1|ITRY_ENTCO). The molecular masses of the two inhibitors determined by means of SDS-PAGE and mass spectrometry are, respectively, 18.5 kDa and 19,710.4 Da (EcCI); 20.2 kDa and 19,813.22 Da (EcTI). Both inhibitors consist of two polypeptide chains and have several isoforms with acidic pIs, ranging from 5 to 6. Towards chymotrypsin, EcCI is a non competitive inhibitor and shows ki of 8 x 10-8 M, while EcTI is a competitive inhibitor with ki of 48 x 10-8 M. Towards trypsin, EcTI shows non competitive inhibition and ki of 2.8 x 10-8 M. EcCI and EcTI show high (93%) and moderate (38%) chymotrypsin inhibition and EcTI was also able to strongly inhibit trypsin (100%). EcCI and EcTI showed high leukocyte elastase inhibition (85 and 75%), respectively, and inhibited pancreatic elastase weakly (about 10%). Neither of them was able to inhibit papain nor bromelain. Both inhibitors are functionally stable under wide temperature range (from 37 to 70 ÂC â EcCI; from 37 to 60 ÂC â EcTI), pH (from 2 to 12) and DTT concentration (from 1 to 10 mM â EcCI; from 1 to 100 mM â EcTI). Both EcCI and EcTI were able to inhibit HT29 colorectal adenocarcinoma cells with IC50 of 35.5 and 20.4 x 10-6 M, respectively. These results clearly indicate that these are molecules with interesting biotechnological features and very promising tools as chemopreventive agents. |
publishDate |
2014 |
dc.date.issued.fl_str_mv |
2014-09-18 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
status_str |
publishedVersion |
format |
doctoralThesis |
dc.identifier.uri.fl_str_mv |
http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=15542 |
url |
http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=15542 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal do Cearà |
dc.publisher.program.fl_str_mv |
Programa de PÃs-GraduaÃÃo em BioquÃmica |
dc.publisher.initials.fl_str_mv |
UFC |
dc.publisher.country.fl_str_mv |
BR |
publisher.none.fl_str_mv |
Universidade Federal do Cearà |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da UFC instname:Universidade Federal do Ceará instacron:UFC |
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Biblioteca Digital de Teses e Dissertações da UFC |
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Biblioteca Digital de Teses e Dissertações da UFC |
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Universidade Federal do Ceará |
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UFC |
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UFC |
repository.name.fl_str_mv |
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repository.mail.fl_str_mv |
mail@mail.com |
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1643295211663654912 |