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