VASCONCELOS, Eliane Silva Araújo de. Proteases do látex de Calotropis procera: purificação, caracterização bioquímica, enzimática e molecular e atividades biológicas. 2013. 140 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2013. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-09-02T11:59:28Z No. of bitstreams: 1 2013_tese_esaraujo.pdf: 2565494 bytes, checksum: 4d162fd198a10a8d88c2c3e0811e739d (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-05T20:30:20Z (GMT) No. of bitstreams: 1 2013_tese_esaraujo.pdf: 2565494 bytes, checksum: 4d162fd198a10a8d88c2c3e0811e739d (MD5) / Made available in DSpace on 2016-09-05T20:30:20Z (GMT). No. of bitstreams: 1 2013_tese_esaraujo.pdf: 2565494 bytes, checksum: 4d162fd198a10a8d88c2c3e0811e739d (MD5) Previous issue date: 2013 / 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 °C 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 têm demonstrado que látex de plantas é uma rica fonte de enzimas com atividades proteolíticas. O isolamento e a caracterização de proteases cisteínicas de látex têm sido recentemente relatados. Neste trabalho nós reportamos a purificação e caracterização de três novas proteases cisteínicas do fluido laticífero de Calotropis procera, bem como sua atividade em ensaios de coagulação plasmática. As três proteases, denominadas CpCP-1, CpCP-2 e CpCP-3 são isoformas de proteases cisteínicas e foram purificadas utilizando dois passos sequenciais de cromatografias de troca iônica 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 sequência de aminoácidos da região N-terminal foi idêntica para as três enzimas, sendo constituída de 30 resíduos de aminoácidos. Análises de sequências revelaram alto nível de identidade (88%) com proteases cisteínicas A atividade proteolítica dessas enzimas foi testada frente a diferentes substratos (Azocaseína, BANA e BApNA) e em diferentes valores de pH e temperatura. As três enzimas foram capazes de degradar Azocaseína e BANA, substratos inespecífico e específico para proteases cisteínicas, respectivamente. CpCP-1 apresentou atividade proteolítica 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 60ºC de temperatura, e o pH ótimo para essas atividades foi 6,0. Análises de Dicroísmo Circular revelaram que a estrutura secundária das proteases era composta de 15,1-19,9% de alfa-hélices e 20,6-21,3% de folhas-beta. Os espectros de desconvolução das proteases mostrou que suas estruturas foram alteradas na presença do agente redutor DTT, sugerindo a presença de pontes dissulfeto na estabilização das estruturas tridimensionais. Em testes biológicos as proteases foram capazes de inibir fortemente a germinação de esporos do fungo Colletotrichum gloesporioides e também exibiram atividade de coagulação plasmática por um mecanismo do tipo trombina.

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.

Made available in DSpace on 2014-12-17T14:16:35Z (GMT). No. of bitstreams: 1 YamaraASM_DISSERT_Parcial.pdf: 1235608 bytes, checksum: 64be8e29311055ebff593313fa2f1681 (MD5) Previous issue date: 2013-07-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocase?na and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The A? chain and B? of fibrinogen were completely cleaved at a concentration of 0.18 ?g/?L of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60?C. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory / A extra??o, caracteriza??o qu?mica e estrutural de uma grande diversidade de compostos derivados de plantas tem sido uma fonte importante de mol?culas bioativas. Diversas proteases t?m sido isoladas no reino vegetal, com in?meras aplica??es farmacol?gicas e biotecnol?gicas. Dentre as proteases isoladas de plantas, est?o as fibrinogenol?ticas, com relevante aplica??o no tratamento de dist?rbios na cascata da coagula??o, al?m do uso em potencial como ferramenta em laborat?rios cl?nicos. Neste trabalho, al?m de avaliar os efeitos do extrato prot?ico de Cnidoscolus urens (L.) Arthur, pertencente ? fam?lia Euphorbiaceae, na cascata de coagula??o, tamb?m se investigou a presen?a de atividade antimicrobiana e caracterizou a atividade proteol?tica detectada neste extrato, tendo como objetivo determinar sua potencial aplica??o farmacol?gica e biotecnol?gica. Desse modo, extratos prot?icos brutos obtidos das folhas de C. urens em tamp?o Tris-HCl 0,05M, NaCl 0,15M, pH 7,5, foram precipitados em diferentes concentra??es de acetona, e avaliados quanto a presen?a de atividade proteol?tica em azocase?na e fibrinog?nio. A fra??o mais ativa (F1.0) nestes testes foi escolhida para realiza??o de avalia??o de atividade biol?gica e caracteriza??o bioqu?mica. As cadeias A? e B? do fibrinog?nio foram completamente clivadas na concentra??o de 0.18 ?g/?L de prote?na da fra??o em 4 minutos. A atividade fibrinogenol?tica apresentou inibi??o total em presen?a de E-64 e parcial em presen?a de EDTA. A fra??o demonstrou atividade coagulante sobre o plasma e reduziu o tempo de tromboplastina parcial ativada, indicando atuar sobre os fatores da via intr?nseca e comum da coagula??o, n?o exercendo efeitos sobre o tempo de protrombina. A atividade fibrinol?tica sobre o co?gulo de plasma foi detectado apenas em SDS-PAGE em concentra??es elevadas da fra??o, e apesar da atividade fibrin(ogen)ol?tica, n?o foi observada atividade defibrinogenante in vivo. Apesar de v?rias proteases de plantas e animais pe?onhentos serem classicamente t?xicas, a frac??o F1.0 n?o expressou atividade hemorr?gica nem hemol?tica. A fra??o F1.0 tamb?m n?o demonstrou atividade antimicrobiana. Na avalia??o da atividade proteol?tica sobre a azocase?na, o pH ?timo de rea??o foi 5.0, e a temperatura ?tima igual a 60?C. A atividade enzim?tica demonstrou ser sens?vel ? presen?a dos sais testados, com inibi??o para todos os compostos. O tensoativo triton n?o influenciou a atividade enzim?tica, por?m o tween-20 e SDS inibiram tal atividade. Em presen?a de agentes redutores ocorreu aumento da atividade enzim?tica, caracter?stica t?pica de enzimas pertencentes ? classe das ciste?no proteases. Diversas bandas prot?icas com atividade proteol?tica foram detectadas em zimograma, na regi?o de elevada massa molecular, que foram inibidas por E-64. Neste trabalho, foi revelado que C. urens apresenta fra??o enriquecida com ciste?no-proteases que apresentam atividade fibrinogenol?tica e procoagulante, que podem ser isoladas, com potencial aplica??o no tratamento de dist?rbios hemorr?gicos, como trombol?tico e em laborat?rio cl?nico / 2020-01-01