Gustavo A. Sandoval

Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: Molecular sequence analysis of its cDNA and biochemical properties

The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.

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.