Padronização de ensaios para dosagem de atividade fosfolipásica em venenos de serpentes

Abstract

Snake venom is a mixture with great molecular diversity that has inter and intraspecific implications and, consequently, leads to a variety of pathophysiological activities and complexity in the production of antivenom. Among this molecular diversity, phospholipases are major hydrolytic enzymes in snake venoms and cause a wide range of pharmacological effects. The present study seeks to standardize assays for the measurement of phospholipase activity for a screening profile aimed at the Toxinology Laboratory of the Centro de Estudos e Pesquisas Biológicas at PUC Goiás through the assays described by Marinetti (1965) and Gutiérrez, Ownby and Odell (1984), beyond to performing a complementary indirect hemolysis plate assay. Both assays were standardized for the conditions of the CEPB Toxinology Laboratory and are commonly characterized by being simple, fast and inexpensive. Substrate standardization revealed concentrations of 2,5 mg/ml and 3,125 mg/ml as ideal for the assays by Marinetti (1965) and Gutiérrez, Ownby and Odell (1984), respectively. Additionally, a venom standardization was performed using the Bothrops moojeni venom as a parameter, with a concentration of up to 0,5 mg/ml considered appropriate for both assays. The complementary indirect hemolysis plaque assay also exhibited similarities between concentrations of 0,5 and 1 mg/ml. Bothrops jararacussu, Bothrops alternatus, Bothrops atrox and Crotalus durissus collilineatus venom samples were tested by standardized assays indicating a high and similar phospholipase activity (general and PLA2) for B. jararacussu and C.d. collilineatus, moderate for B. atrox and low for B. alternatus. Notwithstanding that the treated assays are characterized as less recommended for the measurement of phospholipase activity, we emphasize the importance of these simple spectrophotometric assays for preliminary tests in scientific research, considering the standardization of biochemical assays as fundamental in the area of experimental biology.