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The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A(2) (PLA(2)s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca2+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [I-125]iodinated alpha-bungarotoxin for binding to human alpha 7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L stagnalis, the IC50 being 20 +/- 1.5, 4.3 +/- 0.2, and 10.6 +/- 0.6 mu M, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L stagnalis neurons with IC50 of 11.4 mu M. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA(2)s from snake venoms. Indeed, the protein possessed high PLA(2) activity, which was 1.95 mmol/min mu mol.. Bitanarin is the first described PLA(2) that contains 14 disulphide bonds and the first nAChR blocker possessing PLA2 activity. (C) 2011 Elsevier Ltd. All rights reserved.