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Abstract
In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A2 (PLA2) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA2-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA2-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA2-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA2-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA2s. The phylogenetic analyses showed that BpPLA2-TXI forms a group with other acidic D49 PLA2s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects.
Highlights
Bothrops pauloensis is a venomous snake widely distributed throughout the Brazilian territory, except in the Amazonian region of Brazil
An acidic phospholipase A2 (PLA2) isolated from the venom of B. pauloensis was obtained by four chromatographic procedures in the present work
The Q2 fraction was applied on a reverse phase high performance liquid chromatography (HPLC) C2–C18 μRPC 4.6/100 (GE HealthCare) and the acidic PLA2, named BpPLA2-TXI, was eluted with approximately 80% of the Solvent B (Figure 1D)
Summary
Bothrops pauloensis is a venomous snake widely distributed throughout the Brazilian territory, except in the Amazonian region of Brazil. The main toxins present in B. pauloensis snake venom include metalloproteinases, phospholipases A2, serine proteinases, L-amino acid oxidases, disintegrins, nucleotidases and hyaluronidases, among others [2]. PLA2s are present in snake venoms and are characterized by low molecular mass (13–18 kDa), histidine at the catalytic site, Ca2+ bound at the active site, as well as six conserved disulfide bonds with one or two variable disulfide bonds [7]. These PLA2s can be divided into two groups, I and II, whereby the second is subdivided into two classes, and are present in snake venoms from the Viperidae family.
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