CONICET | Buscador de Institutos y Recursos Humanos

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elseviers archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyrightAuthor´s personal copyIsolation, amino acid sequence and biological characterization of anaspartic-49 phospholipase A2 from Bothrops (Rhinocerophis)ammodytoides venomHerlinda Clement a,1, Vanessa Costa de Oliveira b,1, Fernando Z. Zamudio a, Néstor R. Lago b,Norma A. Valdez-Cruz c, Melisa Bérnard Valle a, Silvia E. Hajos e, Alejandro Alagón a,Lourival D. Possani a, Adolfo R. de Roodt b,d,*a Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Avenida Universidad, 2001,Apartado Postal 510-3, Cuernavaca Morelos 62210, Mexicob Laboratorio de Toxinopatología, Centro de Patología Experimental y Aplicada, Facultad de Medicina, Universidad de Buenos Aires, Argentinac Departamento de Biología Molecular y Biotecnología Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228,México, D.F., CP 04510, Mexicod Instituto Nacional de Producción de Biológicos, A.N.L.I.S. Dr. Carlos G. Malbrán, Ministerio de Salud, Argentinae Cátedra de Inmunología Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentinaa r t i c l e i n f oArticle history:Received 26 May 2012Received in revised form 24 August 2012Accepted 30 August 2012Available online 10 September 2012Keywords:Bothrops ammodytoidesRhinocerophis ammodytoidesPhylogenyPhospholipaseSnakeVenoma b s t r a c tA phospholipase enzyme was separated by chromatography from the venom of the snakeBothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determinedmolecular weight was 13,853.65 Da, and the full primary structure was determined byEdman degradation and mass spectrometry analysis. The enzyme contains 122 aminoacids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13.Sequence comparison with other known secretory PLA2 shows that the enzyme isolatedbelongs to the group II, presenting an aspartic acid residue at position 48 (numbered byconvention as Asp49) of the active site, and accordingly displaying enzymatic activity. Theenzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic tomice. The intravenous LD50 of this phospholipase in CD-1 mice was around 6 mg/g ofmouse body weight (more exactly 117 mg/mouse of 20 g) and the minimal mortal dose(MMD) was estimated to be close to 10 mg/g. In contrast, the LD50 of the venom was circa2 mg/g mouse body weight. Toxicological analyses of the purified enzyme were performedin vitro and in vivo using experimental animals (mice and rats). The enzyme at high dosescaused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction andmuscle tissue alterations with increasing of creatine kinase levels. 2012 Elsevier Ltd. All rights reserved.1. IntroductionBothrops ammodytoides is the most Southern situatedviper in the world. This snake inhabits a geographicalregion that goes from the warm desert regions to the coldPatagonia, in the South of the American Continent. Thisviper was reclassified under the genus Rhinocerophis(Fenwick et al., 2009). Carrasco et al. (2012) proposedcalling this snake as belonging to the genus Rhinocerophis,synonymizing Bothrops, however according to the presentnomenclature accepted it is known by the name of Bothropsammodytoides. It is a small viper that usually does notsurpass 70 cm in length, but can cause the typical problems* Corresponding author. Laboratorio de Toxinopatología, Centro dePatología Experimental y Aplicada, Facultad de Medicina, Universidad deBuenos Aires, Uriburu 950, 5 Piso, CP 1114 CABA, Argentina.E-mail address: aderoodt@gmail.com (A.R. de Roodt).