Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom

• Published in: Biochimie Vol. 89; no. 3; pp. 319 - 328
• Main Authors: Higuchi, D A, Barbosa, C M V, Bincoletto, C, Chagas, J R, Magalhaes, A, Richardson, M, Sanchez, E F, Pesquero, J B, Araujo, R C, Pesquero, J L
• Format: Journal Article
• Language: English
• Published: France 01-03-2007
• Subjects: Amino Acid Sequence; Animals; Anticoagulants - pharmacology; Blood Coagulation - drug effects; Bothrops - metabolism; Calcium - metabolism; Carcinoma, Ehrlich Tumor - chemically induced; Carcinoma, Ehrlich Tumor - enzymology; Carcinoma, Ehrlich Tumor - pathology; Cell Survival - drug effects; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Enzyme-Linked Immunosorbent Assay; Hemoglobins - metabolism; Humans; Isoenzymes - chemistry; Isoenzymes - isolation & purification; Isoenzymes - metabolism; K562 Cells; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Molecular Weight; Phospholipases A - chemistry; Phospholipases A - isolation & purification; Phospholipases A - metabolism; Platelet Aggregation - drug effects; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Snake Venoms - enzymology; Snake Venoms - pharmacology
• ISSN: 0300-9084
• DOI: 10.1016/j.biochi.2006.10.010

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.