Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes

Partial purification and some physicochemical properties of phospholipases A2 from the venom of the bushmaster snake (Lachesis muta)

Screening of the biochemical-pharmacological properties of the crude venom from the snake Lachesis muta indicated the presence of phospholipase A2 (PLA2; 5260 U/mg protein), procoagulant (2630 U/mg protein), platelet aggregating (43 U/mg protein) and caseinolytic activities (6670 U/mg protein). These activities were separated by filtration of the crude venom on Sephacryl S-200. The material containing PLA2 activity was further fractioned by DEAE-cellulose ion exchange chromatography into four active fractions (F-I to F-IV, containing 1.7, 1.2, 0.3, and 0.05 per cent of the crude venom protein, respectively) by stepwise elution with buffers of increasing ionic strength. All fractions presented a molecular weight of approximately 15,000 and isoelectric points in the range pH 4.6-6.0. In addition to their indirect hemolytic activity, the partially purified fractions inhibited platelet aggregation induced either by collagen or thrombin. p-Bromophenacyl bromide-treated fractions lost both phospholipase A2 activity and their inhibitory effect on collagen-induced platelet aggregation

Purification, physicochemical characterization and N-terminal-amino acid sequence of a phospholipase A2 from Bothrops jararaca venom

Snake venoms usually contain multiple molecular forms of phospholipase A2 enzymes (phosphatide acyl hydrolase, E.C. 3.1.1.4; PLA2). Phospholipases A2 induce a wide range of pharmacological effects which may depend or not on the hydrolysis of phospholipids. In this study, a PLA2 from Bothrops jararaca venom was purified to homogeneity by gel filtration on a Sephacryl S-200 column, followed by FPLC reverse-phase chromatography on a Pep-RPC HR 5/5 column (yield 1.63 per cent of venom protein). The PLA2 activity of the fractions was determined by indirect hemolysis using hen's egg yolk lecithin as substrate. The enzyme is an acidic protein with PI 4.5 and an apparent molecular weight of 14,200, as estimated by gel filtration on a Superose 12 FPLC column. Similar properties have been described for PLA2 from other snake venoms. The N-terminal-sequence of the purified protein was NLMQFETMIMXXAGQ. These partial sequence data show a high degree of homology between the B. jararaca PLA2 and the enzymes from other snake venoms as well as bovine pancreatic PLA2

Purification and partial characterization of phospholipase A2 from ascaris.

A fraction with phospholipase A2 activity [EC 3,1,1,4] has been purified from Ascaris worms by a combination of gel filtration on Sephadex G-100 [fine] and ion exchange chromatography on DEAE-Sephacel. The enzyme showed a single band by SDS-polyacrylamide gel electrophoresis and had a molecular weight of about 19,000. The final preparation was purified 46 fold. It has 18% carbohydrate content. Optimum temperature for enzyme activity was 50°C and the optimum pH was 6.0. It has a Km value about 79 mg/L. It could be activated by calcium, manganese and cobalt and it was inhibited by EDTA and iodoacetate. Studying kinetics of phospholipase may be of value in developing a chemotherapeutic agent that inhibits parasite lipid metabolism with consequent inhibition of its vital activities resulting in parasite control

Anticoagulant effect of myotoxic phospholipase A2 isolated from the venom of the snake Bothrops asper (Viperidae)

La fosfolipasa A2 miotoxica del veneno de Bothrops asper (tercipelo) prolonga el tiempo de recalcificación de plasmas de cinco especies de mamíferos. La fosfolipasa A2 hidroliza los fosfolípidos del plasma, en tanto que su acción inhibitoria es revertida cuando se adicionan fosfolípidos plaquetarios. Estas dos observaciones sugieren que la acción anticoagulante se debe a una alteración de los fosfolípidos plasmáticos necesarios para la coagulación. Tanto la actividad enzimática como la anticoagulante se mantuvieron aún después de calentamiento a 95è-C durante 10 min. Pese a su acción anticoagulante in vitro, la fosfolipasa A2 no prolonga el tiempo de coagulación luego de inoculación intravenosa en ratones