Physicoquemical and biologic characterization of phospholipase A2 isolated from Bothrops moojeni venom

Author(s)

Andrana Karla Calgarotto

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2008-03-14

Examining board members

Caroline Ribeiro de Borja Oliveira; Hiroshi Aoyama

• Advisor: Sergio Marangoni

Abstract

Bothrops moojeni is a very important snake species due to its great distribution in the South and Central America besides the clinic alterations caused by the venom. Among the Brazilian species the Bothrops genus is the most numerous and shows the highest registrations related to ophidian accidents. The snake venoms are source of biologically active substances which main components are proteins. Phospholipases A2 (PLA2), proteins present in the snake venom, hydrolyze the sn-2 acyl groups of phospholipids liberating fatty acids and lysophospholipds, besides exhibiting many pharmacological effects. The fosfolipase A2 (PLA2) isoform, named BmTX-I was isolated through RP-HPLC performed on a C18 column. The high degree of purity was confirmed by SDS-PAGE and also by determining the molecular mass (14238.71 Da) in a MALDI TOF mass spectrometry. The kinetic characterization of BmTX-I PLA2 (Asp49) showed that the isoform was highly stable and presented an optimum pH of 8.0 and temperature of 37° C. At different substrate acid 4-nitro-3-(octanoyloxy) benzoic concentrations the BmTX-I showed allosteric behavior. In the absence of Ca2+ and in the presence of some divalent ions such as Mg+2, Mn+2, Cd2+ (at the concentration of 10 mM) the BmTX-I activity significantly decreased. But in the presence of 1mM Ca+2, under the same divalent ions conditions, the isoforms showed a discreet activity. An inhibitory effect of crotalic crotapotins on the activity PLA2 was also demonstrated. The analysis of amino acids composition showed a high content of basic amino acids such as Lys, His and Arg indicating a basic character for the BmTX-I. The presence of 14 cysteine residues suggests the formation of 7 disulfide bridges. N-terminal amino acid sequence revealed a high level of homology between BmTX-I and other Asp49 PLA2s. The neurotoxic effect of the whole venom and of BmTX-I was analyzed at chick biventer cervicis muscle preparation. Our results showed that the blockage of the muscle contraction was lesser when compared with crotalic venoms, which blockage at lower concentrations. Nevertheless it is sure that there was an in vitro neurotoxic action. The complete blockage, as much the whole venom as the BmTX-I, was not accompanied by any significant inhibition of the responses to KCl and to Ach, excepting at higher concentrations of the venom (50 e 100 µg/ml) suggesting the primordial presynaptic action. The tests in vivo with the whole venom and BmTX-I fraction showed the miotoxic effect through the creatine kinase (CK) releases and the inflammatory effect through edema-forming activity and interleukin-6 release. The CK release was similar for both whole venom and BmTX-I, which showed higher liberation one hour after the i.m injection. After eight hours of injection the CK levels were similar to the controls. There was the IL-6 release as well as edema-forming activity both in to the whole venom and BmTX-I. The reprodubility of pharmacological effects, is just possible with the utilization of chemically homogenous fractions that maintain the integrity of biological function. These fractions were obtained with high efficient methodologies as HPLC, through which this we the BmTX-I could be isolated in only one chromatography step, with purity direct by mass spectrometry. These results may be associated with the biological activities, eliminating the subjectivity caused by total venom or impure fractions. This approximation may be applied to the biochemical, structure-function, physiological and pharmacological studies, and it may reveal still unknown mechanisms in the structure-function relationship of PLA2 from the serpents venom (AU)