Biochemical and enzymatic characterization of two basic Asp₄₉ phospholipase A₂ isoforms from Lachesis muta muta (Surucucu) venom

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• 作者：Daniela C.S. Damico ; Sé ; rgio Lilla ; Gilberto de Nucci ; Luis A. Ponce-Soto ; Flá ; via V. Winck ; José ; Camillo Novello and Sé ; rgio Marangoni
• 关键词：Characterization ; Enzyme ; Lachesis muta muta ; Phospholipase A₂ ; Snake venom
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：2005
• 出版时间：2005
• 年：2005
• 卷：1726
• 期：1
• 页码：75-86
• 全文大小：336 K

文摘

Two basic phospholipase A₂ (PLA₂) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 μ-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg₆₅ (LmTX-I) → Pro₆₅ (LmTX-II). The amino acid sequence showed a high degree of homology between PLA₂ isoforms from Lachesis muta muta and other PLA₂ snake venoms. LmTX-I and LmTX-II had PLA₂ activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35–45 °C. Full PLA₂ activity required Ca²⁺ and was respectively inhibited by Cu²⁺ and Zn²⁺ in the presence and absence of Ca²⁺. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P < 0.05) the enzymatic activity of LmTX-I, suggesting that the binding site for crotapotin in this PLA₂ was similar to another in the basic PLA₂ of the crotoxin complex from C. durissus cascavella venom.