摘要
摘要从旅顺产白眉蝮蛇(Glyodius blomhoffi Brevicaudaus,GBB)蛇毒(GBBSV)中首次纯化得到了电泳纯和质谱纯磷脂酶A2(GBB-PLA2)。采用电泳、质谱、等离子体发射光谱、紫外、荧光光谱测试技术对GBB-PLA2进行了系统表征。HPLC-ESI-MS法测得的精确分子量为13 965·07 amu,HPLC-ESI-MS/MS法鉴定GBB-PLA2为江浙蝮蛇和尖吻蝮蛇蛇毒PLA2的同源性蛋白,分别得到4个和1个同源性肽段。凝胶过滤、质谱和SDS-PAGE测试技术结果表明,GBB-PLA2以稳定的单体形式存在。电感耦合等离子体发射光谱分析法(ICP-AES)测得每1个磷脂酶A2含有1个Ca2+离子,Ca2+是PLA2活性所必需的。Ca2+的除去使PLA2活性降低90%,外加Ca2+明显加速PLA2水解底物DPPC的速度。Ca2+起稳定PLA2结构的作用,可使GBB-PLA2的热变性温度提高3℃。
A novel phospholipase A2 （PLA2 ） named GBB-PLA2 was purified and determined by SDS-PAGE and mass spectrometry, from the snake venom of Glyodius blomhoffi Brevicaudaus. Electrophoresis, mass spectrometry, ICP-AES, uv-vis and fluorescence spectroscopy techniques were utilized for the systematic study of GBB-PLA2. The purified protein possessed molecular mass of 13 965.07 ainu. Protein identification of GBBPLA2 was carried out by HPLC-ESI-MS/MS analysis. Four homogeneous peptides and 1 peptide of two PLA2s Agkistrodon halys Pallas and Deinagkisrodon acutus snake venoms were identified from GBB-PLA2. The results from native Sephacryl S-300 gel filtration and MALDI-TOF mass spectrometry indicate that GBB-PLA2 exists as a stable monomeric molecule in aqueous solution. The experimental data from ICP-AES reveal that the purified GBB-PLA2 is a Ca^2＋ bound protein with a calcium stoichiometry of 1：1 （ [ Ca^2＋ ]/[ GBB-PLA2 ] ）. Removal of Ca^2＋ from GBB-PLA2 decreases the hydrolysis activity of PLA2 by 90%, suggesting that Ca^2＋ ions play a critical role for GBB-PLA2. The addition of exogenous Ca^2＋ can accelerate the hydrolysis rate of the substrate DPPC for GBB-PLA2. Ca^2＋ increases the thermostability by 3 ℃ and changes the secondary structure of GBB-PLA2, indicating that the presence of Ca^2＋ helps to maintain the structure integrity of GBB-PLA2.
出处
《应用化学》
CAS
CSCD
北大核心
2009年第3期257-262,共6页
Chinese Journal of Applied Chemistry
基金
国家自然科学基金(30512741)
大连医科大学高科技科研启动经费(20070301)资助项目
作者简介
通讯联系人：孙明忠，男，博士，教授；E-mail：mxs288@gmail．com；研究方向：蛋白质结构与功能
