Fanny Lazo

Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta(Peruvian bushmaster)

A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.

Eficacia experimental de anticuerpos IgY producidos en huevos, contra el veneno de la serpiente peruana Bothrops atrox

Objectives. To develop an immunization protocol in order to produce avian Igy immunoglobulins against Bothrops atrox Peruvian snake venom and to evaluate its neutralizing capacity. Materials and methods. Six Hy Line Brown hens were immunized each two weeks using 500µg/doses of B. atrox venom in a period of two months. Each week, eggs were collected for Igy isolation from yolk using two consecutive steps with caprilic acid and ammonium sulfate. Detection of Igy anti-B. atrox were performed by double immunodiffusion, whereas title and cross-reactivity were analyzed using ELISA and Western Blot technics, respectively. furthermore, letal dose (DL 50) and Medium Effective Dose (DE50) were obtained by Probit analysis. Results. As a result of this protocol, chicken Igy’s were obtained in a concentration of 8,5 ± 1,35 mg/yolk mL. DE50 from avian antivenom was 575 µL/venom mg. Cross-reactivity studies showed Bothrops atrox venom share more commom epitopes with Bothrops brazili (47%) than others Bothrops venoms showing Lachesis muta (19%) and Crotalus durissus (12%) venoms a low crossing reactivity, instead. Conclusions. Using this procedure, we could purify chicken Igy with a neutralizant capacity of B. atrox venom which is comparable to the antivenom of equine origin and demonstrate its capacity as a immunoanalitical tool to evaluate the cross reactivity with others peruvian snakes.

Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom

ABSTRACT An L‐amino acid oxidase from Peruvian Bothrops pictus (Bpic‐LAAO) snake venom was purified using a combination of size‐exclusion and ion‐exchange chromatography. Bpic‐LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ˜65 kDa under reducing conditions and ˜132 kDa in its native form as analyzed by SDS‐PAGE and gel filtration chromatography, respectively. N‐terminal amino acid sequencing showed highly conserved residues in a glutamine‐rich motif related to binding substrate. The enzyme exhibited optimal activity towards L‐Leu at pH 8.5, and like other reported SV‐LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca2+, Mg2+ and Mn2+ did not alter Bpic‐LAAO activity; however, Zn2+ is an inhibitor. Some reagents such as &bgr;‐mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic‐LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic‐LAAO, this enzyme induced edema in mice (MED = 7.8 &mgr;g), and inhibited human platelet aggregation induced by ADP in a dose‐dependent manner and showed antibacterial activity on Gram (+) and Gram (‐) bacteria. Bpic‐LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well‐established monophyletic groups in Viperidae and Elapidae families. Bpic‐LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well‐defined cluster of the Bothrops genus. HighlightsWe present a structure‐function relationships of an L‐amino acid oxidase, Bpic‐LAAO, from Bothrops pictus.Bpic‐LAAO is a dimeric glycoprotein and its sequence was deduced from its cDNA.Bpic‐LAAO reduced the growth of P. aeruginosa, V. cholerae, S. aureus, E. faecalis and E. coli.

Variaciones en las actividades enzimáticas del veneno de la serpiente Bothrops atrox "jergón", de tres zonas geográficas del Perú

RESUMEN Objetivos. Estudiar la variabilidad en la composicion y actividades enzimaticas entre venenos de ejemplares adultos de Bothrops atrox. Materiales y metodos. Se emplearon venenos de serpientes adultas procedentes de Amazonas, Junin y Ucayali. A cada una de las muestras se les realizo el analisis del contenido proteico y del numero de bandas por PAGESDS, asi como las actividades de fosfolipasa A 2 , hemolitica indirecta, amidolitica, coagulante, hemorragica y proteolitica sobre caseina y mediante zimograma; ademas, se hicieron ensayos de inmunodifusion y neutralizacion in vitro con el suero antibotropico polivalente del Instituto Nacional de Salud de Peru. Resultados. Las actividades amidolitica, coagulante, hemorragica, proteolitica mediante zimograma, fosfolipasa A 2 y hemolitica indirecta fueron variables, evidenciandose en las tres ultimas una mayor actividad en los venenos de Amazonas, mientras que en la cantidad de proteina, bandas electroforeticas y actividad proteolitica sobre caseina no se observaron diferencias. Con respecto a las pruebas de neutralizacion, 0,5 dosis del antiveneno fueron suficientes para neutralizar con eficacia (mas del 50%) la actividad coagulante y fosfolipasa A 2 de todas las muestras analizadas. Conclusiones. Algunas propiedades biologicas del veneno de ejemplares adultos de Bothrops atrox de Peru son variables, sin que ello afecte la neutralizacion in vitro por parte del suero antibotropico polivalente sobre las actividades coagulante y fosfolipasa A 2 del veneno. Palabras clave: Venenos de serpiente; Bothrops; Enzimas; Antivenenos (Fuente: DeCS BIREME).

Micrurus spixii (PERUVIAN CORAL SNAKE) VENOM - PRELIMINARY BIOCHEMICAL AND ENZYMATIC CHARACTERIZATION

Micrurus spixii venom was studied after fractionation by Sephadex G-100 SF gel filtration chromatography. Several enzymatic activities and biological effects were investigated in whole venom and fractions. The venom was resolved in four peaks in a range of about 73.2-10.7 kDa molecular weight. Alkaline phosphatase and acetylcholinesterase activities were found in peak I, and procoagulant activity was seen in peak II. Phospholipase A2, hemorrhagic, and proteolytic activities were detected in peak III. A second procoagulant factor and proteinase were present in peak IV. Thrombin-like enzyme and direct hemolytic activities were not found in any assayed samples.