Edith Rodríguez

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.

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.