In the present work, we performed assay-directed fractionation to isolate a vasoactive molecule from the venom of Lasiodora sp. spider.

Preliminary data of our group indicated that it was a low molecular mass compound. Several low molecular mass compounds have been described in the venoms, like free acids, free aminoacids, biogenic amines, neurotransmitters and other organic compounds ( Escoubas et al., 2000, Palma and Nakajima, 2005, Schroeder et al., 2008 and Gomes et al., 2011). Thus, we started the purification with molecular mass filtration followed by reversed-phase chromatography (Fig. 3). NMR analysis of the vasoactive learn more fraction (Table 1; Supplementary data) led us to the identification of ADP as the main compound (Fig. 5).

MS and UV-absorption spectra data corroborated this result. ESI-MS spectrum of our sample showed a compound of 427 Da (Fig. 4A). The molecular mass described for ADP is 427.2 Da. ESI-MS/MS spectrum revealed Quizartinib manufacturer two main fragmented ions as [M + H]+: 348.1 and 136.2 m/z ( Fig. 4B), which corresponds to AMP and adenine, respectively. Additionally, adenine nucleotides have high UV absorption at 254 nm ( Juengling and Kammermeier, 1980), as well as our sample. We believe that the minor quantity of AMP found in the sample occurs due to ADP hydrolysis during reversed-phase chromatography process, as we observed that approximately 12% of an ADP standard sample is converted to AMP inside an HPLC column (data not shown). Nucleotides are composed of a ribose sugar, a nitrogenous base and one or more phosphate groups (Fig. 5). Purine nucleotides have very important roles in nucleic Cyclooxygenase (COX) acid synthesis and energy metabolic pathways. Additionally, they are equally important as extracellular signaling molecules (Burnstock, 2006). Adenosine triphosphate (ATP) and biogenic amines (serotonin and histamine) are currently found in animal venoms. ATP, ADP and AMP have been already described in the venoms of mygalomorph spiders, such as E. californicum, Dugesiella sp. and Aphonopelma sp. ( Schanbacher

et al., 1973, Chan et al., 1975, Odell et al., 1989, Savel-Niemann, 1989 and Weisel-Eichler and Libersat, 2004). The exact role of nucleotides present in the venoms is uncertain. They may participate in envenomation pain and erythema, and help the venom spread by local vasodilation. Nucleotides are possibly products of the high metabolic activity of the venom gland. ATP and ADP regulate vascular homeostasis through the activation of a family of receptors present on the cell surface of platelets, endothelial and vascular smooth muscle cells. Purinergic receptors are P1 (AMP and adenosine) and P2 (ATP and ADP) types. P2 receptor subtypes are P2X ionotropic ligand-gated ion channel receptors and P2Y metabotropic G protein-coupled receptors (Korchazhkina et al., 1999, Burnstock, 2007 and Dodbiba et al., 2010). Purine nucleotides cause vasodilation by action on endothelial cell receptors (Burnstock, 2002 and Burnstock, 2008).