Department of Biochemistry, Institute of Biology (IB), Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil.
Biomed Res Int. 2013;2013:612649. doi: 10.1155/2013/612649. Epub 2012 Dec 30.
Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25-37°C. Maximum PLA2 activity required Ca(2+) and in the presence of Cd(2+), Zn(2+), Mn(2+), and Mg(2+) it was reduced in the presence or absence of Ca(2+). Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.
Bbil-TX 是一种 PLA2，从两头蛇蛇毒中经过一次反相高效液相色谱（RP-HPLC）纯化，使用 μ-Bondapak C-18 柱。通过 Q-Tof Ultima API ESI/MS（TOF MS 模式）质谱法确认其分子量为 14243.8 Da。然后，获得的部分蛋白质序列被提交给 BLASTp，搜索范围仅限于蛇类的 PLA2，并与其他 PLA2 相比显示出高同源性值。PLA2 活性在存在合成底物的情况下表现出来，并显示出最小的 S 型行为，在 pH 8.0 和 25-37°C 时达到最大活性。最大 PLA2 活性需要 Ca(2+)，并且在存在 Cd(2+)、Zn(2+)、Mn(2+)和 Mg(2+)的情况下，无论是否存在 Ca(2+)，其活性都会降低。响尾蛇毒中的 Crotapotin 和白尾鹿血清中的抗出血因子 DA2-II 在最佳条件下显著抑制酶活性。Bbil-TX 在小鼠中引起肌肉坏死。该馏分在肌管和成肌细胞（C2C12）中没有表现出显著的细胞毒性活性。研究了从两头蛇蛇毒中分离的 Bbil-TX 在小鼠血清中引起的炎症事件。血管通透性增加以及 TNF-a、IL-6 和 IL-1 的水平增加。由于 Bbil-TX 发挥更强的促炎作用，因此磷脂水解可能与这些现象有关。
