Laboratorio de Química Biológica, Departamento de Bioquímica, Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste (UNNE), Av. Libertad 5470, Campus Universitario, CP 3400, Corrientes, Argentina.
Toxicon. 2010 Aug 1;56(1):64-74. doi: 10.1016/j.toxicon.2010.02.031. Epub 2010 Mar 21.
An acidic protein with phospholipase A(2) activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column. A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A(2) in correspondence with Asp49 PLA(2) group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA(2) revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 microg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 microg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) "de novo amino acid sequencing", also provides more database about the small group of the non-myotoxic PLA(2)s isolated up to the present.
一种具有磷脂酶 A(2)活性的酸性蛋白，通过两步色谱法从阿根廷东北部毒蛇 Bothrops alternatus 的毒液中纯化到均一性：常规的 Sephadex G-75 凝胶过滤和 C18 HPLC 柱反相。质谱法测定分子量为 14185.48 Da，显示同源二聚体构象。动力学测定表明该酶具有催化活性的磷脂酶 A(2)与 Asp49 PLA(2)基团相对应。该酶命名为 Ba SpII RP4，含有 121 个氨基酸残基的氨基酸组成和计算理论 pI 值为 4.88。与其他 Bothrops PLA(2)的氨基酸序列比对显示高度同源序列（90-56%）。Ba SpII RP4 肌肉纤维的最大剂量为 100μg i.m. 注射途径时没有肌毒性，最高剂量 200μg 时 i.p. 注射也没有致死反应。这种毒素产生轻微的生物学活性，如足肿胀炎症和凝血时间延迟，尽管 Ba SpII RP4 表现出催化活性。通过 Micromass（英国曼彻斯特）的四重飞行时间（Q-TOF）混合质谱仪 Q-TOF Ultima 测定的一级氨基酸序列，该质谱仪配备了纳米 Zspray 源，以正离子模式运行，串联质谱，ESI/MS 质谱（TOF MS 模式）“从头氨基酸测序”，还提供了更多关于目前为止分离的非肌毒性 PLA(2)s 的小群体的数据库。
