nÃo hà / Studies have shown that latex of plants is a rich source of enzymes with proteolytic activities. Isolation and characterization of cysteine proteases of latex have recently been reported. In this work we report the purification and characterization of three new cysteine proteases of laticifer fluid of Calotropis procera, as well as its activity in plasma coagulation assays. The three proteases, termed CpCP-1, 2-CpCP and CpCP-3 are isoforms of cysteine proteases and were purified using two sequential steps of ion exchange chromatography on CM-Sepharose and Resource S columns, coupled to FPLC system. Their molecular masses were determined by ESI-Q-TOF mass spectrometry: CPCP-1 had mass = 26.213, CPCP-2 = 26.133 and CPCP-3 = 25.086 Da. The amino acid sequences of the N-terminal region was identical for all three enzymes, being composed of 30 amino acid residues. Analysis revealed high sequence identity with others cysteine proteases. The proteolytic activity of these enzymes was tested against different substrates (azocasein, BANA and BApNA) and at different pH and temperature. The three enzymes are capable of degrading azocasein and BANA, substrates nonspecific and specific for cysteine proteases, respectively. CPCP-1 showed proteolytic activity twice that CPCP-3, and this, a little bigger than CPCP-2. Enzymes maintained 60-80% of their activities even when tested at 60 ÂC temperature, and the optimum pH for these activities was 6.0. Circular Dichroism Analysis showed that the secondary structure of the proteases was composed of 15.1 to 19.9% of alpha-helices and 20.6 to 21.3% of beta-sheets. The spectra deconvolution of proteases showed that their structures were altered in the presence of the reducing agent DTT, suggesting the presence of disulfide bridges stabilizing the three dimensional structures. In biological tests proteases were able to strongly inhibit the germination of spores of the fungus Colletotrichum gloeosporioides and also exhibited plasma coagulation activity by thrombin-like mechanism. / Estudos tÃm demonstrado que lÃtex de plantas à uma rica fonte de enzimas com atividades proteolÃticas. O isolamento e a caracterizaÃÃo de proteases cisteÃnicas de lÃtex tÃm sido recentemente relatados. Neste trabalho nÃs reportamos a purificaÃÃo e caracterizaÃÃo de trÃs novas proteases cisteÃnicas do fluido laticÃfero de Calotropis procera, bem como sua atividade em ensaios de coagulaÃÃo plasmÃtica. As trÃs proteases, denominadas CpCP-1, CpCP-2 e CpCP-3 sÃo isoformas de proteases cisteÃnicas e foram purificadas utilizando dois passos sequenciais de cromatografias de troca iÃnica em colunas de CM-Sepharose e Resource S, acoplada a sistema FPLC. Suas massas moleculares foram determinadas por espectrometria de massas em aparelho do tipo ESI-Q-TOF, onde: CpCP-1 apresentou massa=26,213, CpCP-2=26,133 e CpCP-3=25,086. A sequÃncia de aminoÃcidos da regiÃo N-terminal foi idÃntica para as trÃs enzimas, sendo constituÃda de 30 resÃduos de aminoÃcidos. AnÃlises de sequÃncias revelaram alto nÃvel de identidade (88%) com proteases cisteÃnicas A atividade proteolÃtica dessas enzimas foi testada frente a diferentes substratos (AzocaseÃna, BANA e BApNA) e em diferentes valores de pH e temperatura. As trÃs enzimas foram capazes de degradar AzocaseÃna e BANA, substratos inespecÃfico e especÃfico para proteases cisteÃnicas, respectivamente. CpCP-1 apresentou atividade proteolÃtica duas vezes maior que CpCP-3, e esta, um pouco maior que CpCP-2. As enzimas mantiveram 60-80% de suas atividades mesmo quando ensaiadas a 60ÂC de temperatura, e o pH Ãtimo para essas atividades foi 6,0. AnÃlises de DicroÃsmo Circular revelaram que a estrutura secundÃria das proteases era composta de 15,1-19,9% de alfa-hÃlices e 20,6-21,3% de folhas-beta. Os espectros de desconvoluÃÃo das proteases mostrou que suas estruturas foram alteradas na presenÃa do agente redutor DTT, sugerindo a presenÃa de pontes dissulfeto na estabilizaÃÃo das estruturas tridimensionais. Em testes biolÃgicos as proteases foram capazes de inibir fortemente a germinaÃÃo de esporos do fungo Colletotrichum gloesporioides e tambÃm exibiram atividade de coagulaÃÃo plasmÃtica por um mecanismo do tipo trombina.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:10003 |
| Date | 07 March 2013 |
| Creators | Eliane Silva AraÃjo de Vasconcelos |
| Contributors | MÃrcio Viana Ramos, ClÃverson Diniz Teixeira de Freitas, Ana Cristina de Oliveira Monteiro Moreira, Renato de Azevedo Moreira, Ana Lucia Ponte Freitas |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em BioquÃmica, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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