title: Isolation, amino acid sequence and biological characterization of an "aspartic-49" phospholipase A₂ from Bothrops (Rhinocerophis) ammodytoides venom.
abstract: A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids 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 isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD₅₀ of this phospholipase in CD-1 mice was around 6 μg/g of mouse body weight (more exactly 117 μg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 μg/g. In contrast, the LD₅₀ of the venom was circa 2 μg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels.
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Apartado Postal 510-3, Cuernavaca Morelos 62210, Mexico.
authors.forename: Herlinda
authors.initials: H
authors.lastname: Clement
authors.forename: Vanessa
authors.initials: V
authors.lastname: Costa de Oliveira
authors.forename: Fernando Z
authors.initials: FZ
authors.lastname: Zamudio
authors.forename: Néstor R
authors.initials: NR
authors.lastname: Lago
authors.forename: Norma A
authors.initials: NA
authors.lastname: Valdez-Cruz
authors.forename: Melisa
authors.initials: M
authors.lastname: Bérnard Valle
authors.forename: Silvia E
authors.initials: SE
authors.lastname: Hajos
authors.forename: Alejandro
authors.initials: A
authors.lastname: Alagón
authors.forename: Lourival D
authors.initials: LD
authors.lastname: Possani
authors.forename: Adolfo R
authors.initials: AR
authors.lastname: de Roodt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D003402:Creatine Kinase / Q000097:blood; D003435:Crotalid Venoms / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D010802:Phylogeny; D051381:Rats; D017208:Rats, Wistar
chemical_list: D003435:Crotalid Venoms; D003402:Creatine Kinase; D054467:Phospholipases A2