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Orientadores: Sergio Marangoni, Luis Alberto Ponce Soto Dissertação ( mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Os venenos de serpentes contêm uma variedade de proteínas que são estudadas no mundo pela importância biológica e farmacológica. Dentro de sua complexa composição, o veneno apresenta enzimas proteases, como as metaloproteases e serinoproteases que estão envolvidas em distúrbios hemostáticos e coagulantes, interferindo na agregação plaquetária. O presente trabalho teve como objetivo purificar, caracterizar bioquimicamente e avaliar as atividades farmacológicas de uma serinoprotease com atividade coagulante, do veneno de Bothrops barnetti nomeada TLBbar. A purificação envolveu dois passos cromatográficos, exclusão molecular em Sephadex G-75 e HPLC de fase reversa em coluna analítica Supelco C8, obtendo-se um alto grau de pureza e homogeneidade, revelado com SDSPAGE e com Mr ~ 28,5 kDa. TLBbar mostrou homlogia sequêncial com outras serinoproteases de veneno de serpentes, evidenciado através de espectrometria de massas MS/MS, como a trombina-like denominada Calobin, isolada e caracterizada a partir de Agkistrodon caliginosus (Korean Viper), Crotalase (Crotalus adamanteus), flavoxobin (Trimeresurus flavoviridis) entre outras. Observa-se na homologia que as serinoproteases, com as quais foi comparada a TLBbar, apresentam os aminoácidos conservados do sítio catalítico (His57, Asp102, Ser195), já na sequencia da TLBbar demonstra-se a presença do aminoácido histidina na posição 57 da tríada catalítica. Este resultado em conjunto com as atividades enzimáticas e biológicas, confirmam que TLBbar pertence ao grupo de enzimas serinoproteases com atividade trombina-like. A enzima apresenta sua maior atividade proteolítica a 37oC e em pH 8, sendo essa alterada na presença de íons divalentes por interações eletrostáticas entre enzima e substrato. TLBbar apresenta um comportamento michaeliano segundo os estudos cinéticos realizados com o substrato BApNA, revelando um Vmax e Km de 0,42nmol/min e 0,433mM, respectivamente. Em presença de heparina mantem 80% de sua atividade, e apresenta relativa estabilidade frente a SBT-I e agentes quelantes como EDTA e EGTA. TLBbar tem atividade coagulante sobre fibrinogênio bovino (atividade fibrigenolítica), formando um coágulo semi-rígido, pois não ativa o fator XIII da cascata de coagulação como faz a trombina que resulta na formação de um coágulo consistente. Sua atividade fibrigenolítica foi evidenciada pela liberação dos fibrinopeptídios A, pela degradação da cadeia A? do fibrinogênio, a qual foi inibida por PMSF e Leupeptina, confirmando sua classificação no grupo das serinoproteases. A eficiência desta atividade fibrigenolítica foi observada entre 30 oC e 40oC durante 2 horas de incubação e alterada em presença de íons divalentes como cálcio, magnésio e bário, ressaltando a maior atividade fibrigenolítica com o cálcio. A enzima purificada TLBbar foi capaz de agregar plaquetas e em concentração de 2,5?g evidenciou comportamento semelhante à trombina. Interessante que TLBbar estimula a agregação plaquetária assim como também retarda dita atividade no tempo quando comparado com o controle (trombina). Este fato é devido a sua mudança de conformação estrutural que corresponde à ativação plaquetaria a qual ocorre com mais lentidão (nos primeiros minutos), em comparação a transformação causada pela trombina, tendo como conseqüência a liberação mais lenta de agentes como ADP e tromboxanos A2, necessários para alcançar a adesão plaquetária. A serinoprotease TLBbar não apresenta toxicidade, pois não possui atividades edematogênica, hemorrágica e miotóxica in vivo. Por outro lado, TLBbar evidencia capacidade de dissolver o coágulo de fibrina gerado pela trombina após 36 horas de incubação a 37oC, esta atividade foi inibida com PMSF nas mesmas condições, estes dados sugerem a presença de atividade fibrinolítica Abstract: The snake venoms contain variety of proteins and are studied in the world for biological and pharmacological importance, within their complex composition of the venom protease enzymes present, as metalloproteases and serineproteases that cause hemostatic disorders, coagulantes and platelet aggregation. The present dissertation aimed to purify, biochemically characterize and evaluate the pharmacological activities of a serine protease with coagulant activity named TLBbar from Bothrops barnetti. Its purification involved two chromatographic steps, molecular exclusion in Sephadex G-75 and reversedphase HPLC with a Supelco C8 analytical column, obtaining high purity and homogeneity revealed by SDS-PAGE with Mr ~ 28.5 kDa. The enzyme has its highest proteolytic activity at 37 °C and at pH 8, which is altered in the presence of divalent ions by electrostatic interactions between enzyme and substrate. TLBbar presents a Michaelis-Menten behavior according the kinetic studies revealing a Km and Vmax of 0.433 mM and 0.42 nmol/min, respectively. TLBbar in the presence of heparin maintains its activity around 80%, although has relative stability compared with SBT-I and chelating agents such as EDTA and EGTA. TLBbar has coagulant activity on bovine fibrinogen, forming a clot semi-rigid, different the action of thrombin that results in the formation of a clot consistent. Its fibrigenolytic activity was evidenced by the release of fibrinopeptides A, by degradation of fibrinogen A? chain, which was inhibited by PMSF and Leupeptin, confirming its classification in the group of serineproteases. The efficiency of this activity was observed between 30 and 40 °C during 2 hours of incubation and changed in the presence of divalents ions such as calcium, magnesium and barium, brings out the greatest fibrigenolytic activity with calcium. The purified enzyme TLBbar was capable of aggregating platelets; 2.5?g of concentration evidence similar behavior to thrombin, TLBbar interesting stimulates platelet aggregation as well as delay time in such activity when compared with control (thrombin). We suggest that structural process of platelet activation occurs over a longer period of time with TLBbar as a result of release of ADP and thromboxane A2 agents that happen that occur with less speed to achieve platelet adhesion compared with thrombin. This enzyme not showed edematogenic-activity when compared with the activity caused by B. Barnetti snake venom, did not induce hemorrhagic nor myotoxic effect in vivo. On the other hand, TLBbar had proteolytic activity similar to thrombin, proven in the fibrigenolytic activity on bovine fibrinogen as measured in the release of fibrinopeptides in times of release similar to the control, the proteolytic activity was also evidenced by the ability to dissolve fibrin clot generated by Thrombin after 36 hours of incubation at 37 °C, the activity was inhibited with PMSF under the same conditions, these data suggest the presence of fibrinolytic activity Mestrado Bioquímica Mestre em Biologia Funcional e Molecular

12 páginas, 7 figuras., Snake species within the Bothrops complex (sensu lato) are of medical relevance in Latin America, but knowledge on their venom characteristics is limited, or even unavailable, for some taxa. Perú harbors 17 species of pit vipers, within the genera Bothrops, Bothriechis, Bothrocophias, Porthidium, Crotalus, and Lachesis. This study compared the venoms of twelve species, through chromatographic and electrophoretic profiles, as well as proteolytic and phospholipase A2 (PLA2) activities. Also, proteomic profiles were analyzed for nine of the venoms using a shotgun approach. Results unveiled conspicuous differences in the expression of venom PLA2s among species, six of them presenting scarce levels as judged by RP-HPLC profiles. Since most species within the bothropoid lineage possess venoms with high to intermediate abundances of this protein family, our findings suggest the existence of a phenotypic duality in the expression of venom PLA2s within the Bothrops (sensu lato) complex. Bothrops barnetti and Bothrocophias andianus venoms, very scarce in PLA2s, were shown to lack significant myotoxic activity, highlighting that the observed variability in PLA2 expression bears toxicological correlations with effects attributed to these proteins. Finally, an attempt to identify phylogenetic relationships of bothropoid species from Perú presenting low- or high-PLA2 venom phenotypes showed an interspersed pattern, thus precluding a simple phylogenetic interpretation of this venom compositional dichotomy., We thank Instituto Nacional de Salud (Perú) for providing venoms through an agreement signed with Universidad Peruana Cayetano Heredia. Support to the Proteomics Laboratory of Instituto Clodomiro Picado by Vicerrectoría de Investigación, Universidad de Costa Rica, is also gratefully acknowledged.

La presente investigación se ha realizado en el Laboratorio de Química Biológica de la Escuela Profesional y Académica de Biología, de la Universidad Nacional de San Agustín – Arequipa y en colaboración con el laboratorio de Química de Proteínas, de la Universidad Católica Santa María; desde agosto a diciembre del año 2019. En la presente investigación, se ha logrado purificar y caracterizar bioquímicamente una fosfolipasa A2 y una homóloga K49 a partir del veneno total de Bothrops barnetti de procedencia peruana. En la purificación de ambas proteínas la fosfolipasa A2 y la fosfolipasa A2 homóloga K49, se ha empleado un paso cromatográfico a través de una cromatografía convencional de interacción hidrofóbica, donde se obtuvo nueve fracciones: denominadas del 1 al 9 respectivamente. La fracción 5 fue caracterizada bioquímicamente como una fosfolipasa A2 al medir su actividad enzimática frente a un sustrato cromogénico altamente específico como es el 4-nitro-3 (octanoiloxi) acido benzoico a pH de 7,9. De otro lado se caracterizó estructuralmente una fosfolipasa A2 homóloga K49 desprovista completamente de actividad catalítica. La electroforesis bidimensional (2D), reveló el carácter de 5 fracciones denominadas como P101, P102, P103, P104 y P105 respectivamente por poseer una masa aproximada de 14 kDa y un pI en alrededor de 7.6 a 8.5 lo que evidenciaría la presencia de fosfolipasas A2 y/o homólogas K49, a partir del veneno total desde una perspectiva de aproximación proteómica. La secuencia de aminoácidos fue determinada vía espectrometría de masas ESI-S/MS y se caracterizaron dos toxinas una fosfolipasa A2 y una fosfolipasa A2 homólogaK49. Los estudios de homología secuencial utilizando las bases de datos desde su identificador con el programa MASCOT y la base de datos del Swiss prot, nos permitió confirmar la deducción de la cadena polipeptídica tratándose de estas 2 proteínas estudiadas. Desde la perspectiva farmacológica se exploraron las actividades edematogénicas en el experimento edema de pata, en el que quedó claramente evidenciado su efecto pro- inflamatorio de ambas toxinas, donde la actividad catalítica podría ser o no importante. Hecho que fue confirmado con el dosaje de los niveles de IL-6 los cuales mostraron ser tiempo-dependientes como un efecto local. Tesis

Initial association of platelets after vascular injury is mediated by glycoprotein (GP)Ib-IX-V binding to von Willebrand factor (vWf) immobilized on exposed collagens and eventually leads to thrombus formation. This article provides data about a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), purified from the venom of Bothrops barnetti. This Data in Brief manuscript complements the main research article by providing additional data of the biochemical characterization of Bar-I 10.1016/j.bbagen.2015.12.021 [1].

Las Fosfolipasas A2 (PLA2) son enzimas que intervienen en señales de transducción, activando distintos procesos como la contracción de músculo liso, activación de la cascada de inflamación e intervención en procesos digestivos del intestino; procesando lípidos para su posterior absorción. Dentro del grupo de fosfolipasas existe un subgrupo conocido como “PLA2 homólogas”, las cuales no ejercen actividad catalítica debido a la mutación de un aminoácido en el sitio activo; pero aunque estas toxinas carecen de acción lipolitica, producen efectos biológicos similares a sus homólogas catalíticamente activas, entre los cuales se destacan la miotoxicidad e inflamación.En la presente investigación se buscó encontrar una PLA2 homóloga K49 en el veneno de Bothrops barnetti peruana; mediante una columna de interacción hidrofóbica se obtuvieron diversas enzimas, las cuales por análisis de actividad pro-inflamatoria y electroforesis SDS-PAGE fue posible aislar una muestra de aproximadamente 14 kDa con actividad fosfolipásica en relación al resto, la cual se denominó BbTX-II. Mediante espectrometría de masas se determinó la masa molecular promedio de 14242 Da. y se realizó una caracterización de la estructura primaria, obteniendo la secuencia de 3 péptidos; finalmente se realizó un estudio de homología secuencial determinando que la muestra es una PLA2 homóloga K49. Paralelamente se realizó una caracterización biológica de la muestra para determinar el efecto miotóxico; evaluando los mediadores envueltos en la migración leucocitaria y en el efecto inflamatorio (IL-1, IL-6 y TNFα), observando un aumento de los mediadores dentro de las primeras 3 horas; determinando así un efecto local de la muestra

This work used eleven Peruvian snake venoms (Bothrops andianus, Bothrops atrox, Bothrops barnetti, Bothrops castelnaudi, Bothriopsis chloromelas, Bothrocophias microphthalmus, Bothrops neuwiedi, Bothriopsis oligolepis, Bothriopsis peruviana, Bothrops pictus and Bothriopsis taeniata) to perform in vitro experimentation and determine its main characteristics. Hyaluronidase (HYAL), phospholipase A2 (PLA2), snake venom metalloproteinase (SVMP), snake venom serine protease (SVSP) and l-amino acid oxidase (LAAO) activities; toxicity by cell viability assays using MGSO3, VERO and HeLa cell lineages; and crossed immunoreactivity with Peruvian (PAV) and Brazilian (BAV) antibothropic polyvalent antivenoms, through ELISA and Western Blotting assays, were determined. Results show that the activities tested in this study were not similar amongst the venoms and each species present their own peculiarities, highlighting the diversity within Bothrops complex. All venoms were capable of reducing cell viability of all tested lineages. It was also demonstrated the crossed recognition of all tested venoms by both antivenoms.

We report the comparative proteomic characterization of the venoms of Bothrops atrox, B. barnetti and B. pictus. The venoms were subjected to RP-HPLC and the resulting fractions analyzed by SDS-PAGE. The proteins were cut from the gels, digested with trypsin and identified via peptide mass fingerprint and manual sequencing of selected peptides by MALDI-TOF/TOF mass spectrometry. Around 20-25 proteins were identified belonging to only 6-7 protein families. Metalloproteinases of the classes P-I and P-III were the most abundant proteins in all venoms (58-74% based on peak area A214 nm), followed by phospholipases-A(2) (6.4-14%), disintegrins (3.2-9%) and serine proteinases (7-11%), and some of these proteins occurred in several isoforms. In contrast cysteine-rich secretory proteins and L-amino acid oxidases appeared only as single isoforms and were found only in B. atrox and B. barnetti. C-type lectins were also detected in all venoms but at low levels (~ 5%). Furthermore, the venoms contain variable numbers of peptides (

A glycoprotein from the venom of Bothrops barnetti snake , was purified using gel filtration and ionic exchange chromatography . This protein showed 48 kDa as molecular weight by PAGE-SDS under nonreducing conditions being its carbohydrate content 48% of the total mass protein. This enzyme had activity either Benzoil Arginil-p-Nitroanilide (BApNA) and human citrated plasma. In addition this enzyme was strongly inhibited by PMSF and tripsin soybean inhibitor indicating that it is a seninoproeinase Se ha purificado una glicoproteína del veneno de la serpiente peruana Bothrops barnetti mediante tres pasos cromatográficos que involucran exclusión molecular e intercambio iónico. Mediante PAGE-SDS se determinó que la enzima posee un peso de 48 kDa bajo condiciones no reductoras. El contenido de carbohidratos representa el 48% de la masa total. La enzima presentó actividad sobre el sustrato Benzoil Arginil p Nitroanilida (BApNA) y plasma humano citratado. La enzima fue fuertemente inhibida por el PMSF y el inhibidor de tripsina de soya indicando que se trata de una serinoproteasa.

Se ha purificado una glicoproteína del veneno de la serpiente peruana Bothrops barnetti mediante tres pasos cromatográficos que involucran exclusión molecular e intercambio iónico. Mediante PAGE-SDS se determinó que la enzima posee un peso de 48 kDa bajo condiciones no reductoras. El contenido de carbohidratos representa el 48% de la masa total. La enzima presentó actividad sobre el sustrato Benzoil Arginil p Nitroanilida (BApNA) y plasma humano citratado. La enzima fue fuertemente inhibida por el PMSF y el inhibidor de tripsina de soya indicando que se trata de una serinoproteasa., A glycoprotein from the venom of Bothrops barnetti snake , was purified using gel filtration and ionic exchange chromatography . This protein showed 48 kDa as molecular weight by PAGE-SDS under nonreducing conditions being its carbohydrate content 48% of the total mass protein. This enzyme had activity either Benzoil Arginil-p-Nitroanilide (BApNA) and human citrated plasma. In addition this enzyme was strongly inhibited by PMSF and tripsin soybean inhibitor indicating that it is a seninoproeinase

Se ha aislado una enzima similar a trombina, barnetobina, del veneno de la serpiente peruana Bothrops barnetti, mediante dos pasos cromatográficos sobre CM Sephadex C-50 y Sephadex G-100, en ambos casos utilizando acetato de amonio 0,05 M a pH 5,0. La enzima fue purificada 45 veces con un rendimiento de 14% y por PAGE-SDS se obtuvo una sola banda proteica de 52 kDa en condiciones reductoras con 2β-mercaptoetanol y de 48 kDa en condiciones no reductoras, determinαndose que la enzima consta de una sola cadena polipeptνdica con al menos un enlace disulfuro. El tratamiento con N- glicosidasa (PNGasa F) determinσ que es una glicoproteνna, con un 45% de contenido total de carbohidratos. La enzima mostró tener actividad coagulante sobre plasma citratado y fibrinógeno. También mostró actividad amidásica sobre BApNA y Chromozym TH. La potencia coagulante sobre fibrinógeno bovino fue equivalente a 131 unidades NIH de trombina/mg. La enzima es inhibida por PMSF y por el inhibidor de tripsina de soya calificándola como una serinoproteasa; el pH óptimo para la actividad amidolítica fue de 8,0 y es estable hasta los 40°C. Se demostró mediante inmunoelectroforesis e inmunodifusión la antigenicidad de la enzima frente al suero antibotrópico polivalente, así como su neutralización probada sobre plasma citratado (Dosis eficaz: 250 µl de antiveneno/ mg de enzima). A thrombin- like enzyme, barnetobin, was purified from Bothrops barnetti, peruvian snake venom using CM Sephadex C-50 followed by Sephadex G-100, in both two cases with 0,05 M amonium acetate buffer pH 5,0. The enzyme was purified 45 fold with 14% of yield and the PAGE-SDS showed only protein band of 52 kDa under reducing condition with 2β- mercaptoetanol and 48 kDa under non reducing condition indicating that the enzyme has a single polypeptide chain with disulfide bond. The PNGase treatment showed that it is a basic glycoprotein containing 45% total carbohydrates. The enzyme has coagulant activity on fibrinogen and citrated plasma and amidolytic activity on BApNA and Chromozym TH. The coagulant potency was equivalent to 131 NIH thrombin Units/ mg. In addition the enzyme is inhibited by PMSF and soybean trypsin inhibitor suggesting that is a serine proteinase. The enzyme had optimal amidolytic activity pH was 8,0 and is stable until 40 ºC. The antigenicity and neutralization of enzyme was demonstrated by inmunodiffusion and inmunoelectrophoresis with the polyvalent antibothropic serum on citrated plasma (Effective Doses: 250 µl of antivenom/ mg of enzyme).