高等学校化学学报 ›› 2009, Vol. 30 ›› Issue (9): 1773.

• 研究论文 • 上一篇    下一篇

金属离子对蛇毒蛋白生物活性及结构效应的影响

刘淑清1, 郭春梅1,2, 徐跃飞1, 孙明忠2, 辛毅2, 唐建武3   

  1. 1. 大连医科大学生物化学与分子生物学教研室,
    2. 基础医学院生物技术系,
    3. 病理学教研室, 大连 116044
  • 收稿日期:2009-01-09 出版日期:2009-09-10 发布日期:2009-09-10
  • 通讯作者: 孙明忠, 男, 博士, 教授, 主要从事生物活性物质结构与功能和蛋白质组学研究. E-mail: mxs288@gmail.com; 刘淑清, 女, 博士, 教授, 主要从事生物化学和疾病蛋白质组学研究. E-mail: lsqsmz@gmail.com
  • 基金资助:

    国家自然科学基金(批准号: 30572098, 30772468)和大连市科技厅项目(批准号: 2008J22JH014)资助.

Effect of Metal Ions on the Biological Activity and Structural Information of Snake Venom Proteins

LIU Shu-Qing1*, GUO Chun-Mei1,2, XU Yue-Fei1, SUN Ming-Zhong2*, XIN Yi2, TANG Jian-Wu3   

  1. 1. Department of Biochemistry and Molecular Biology,
    2. Department of Biotechnology, College of Basic Medical Science,
    3. Department of Pathology, Dalian Medical University, Dalian 116044, China
  • Received:2009-01-09 Online:2009-09-10 Published:2009-09-10
  • Contact: LIU Shu-Qing. E-mail: mxs288@gmail.com; SUN Ming-Zhong. E-mail: lsqsmz@gmail.com
  • Supported by:

    国家自然科学基金(批准号: 30572098, 30772468)和大连市科技厅项目(批准号: 2008J22JH014)资助.

摘要:

从旅顺产白眉蝮蛇(Gloydius blomhoffii brevicaudus, GBB) 蛇毒中纯化得到了3种电泳和质谱纯蛋白活性组分, 其酶解肽段采用高效液相色谱-电喷雾串联质谱(HPLC-nESI-MS/MS)进行序列测定, 与其它同源性蛇毒蛋白氨基酸序列比对发现, 3种蛋白为新的蛇毒磷脂酶A2、类凝血酶和金属蛋白酶, 分别将其命名为GBB-bPLA2, GBB-TLE和GBB-MP. 电感耦合等离子体发射光谱法(ICP-AES) 测得每个GBB-bPLA2和GBB-MP中含有一个Ca2+; 每个GBB-TLE中含有2个Zn2+. Ca2+可分别使GBB-bPLA2和GBB-MP的荧光发射波长向短波方向移动2.0和1.6 nm, 使二者的荧光发射强度提高14.0%和11.0%; Ca2+的存在可显著提高二者的热稳定性, 使GBB-bPLA2和GBB-MP的热变性温度分别提高1.5和2.0 ℃. Zn2+使GBB-TLE的荧光发射强度增高4.3%, 但对GBB-TLE的酯酶水解活性、荧光发射波长和热变性温度无显著影响. 金属离子的存在能够不同程度地影响蛇毒蛋白结构热稳定性, 但对蛇毒蛋白生物活性的作用则不同.

关键词: 蛇毒蛋白; 金属离子; 活性

Abstract:

Three bioactive proteins of the snake venom of Gloydius blomhoffii brevicaudus(GBB) were for the first time purified to be homogenous, as determined by both SDS-PAGE and mass spectrometry. HPLC-nESI-MS/MS approach was carried out to get the amino acid sequences of the tryptic-digested peptides. On the basis of the sequencing comparisons of the three proteins with other known snake venom protein homologue, those proteins are identified as three novel basic phospholipase A2(bPLA2), metalloproteinase(MP) and thrombin-like enzyme(TLE), which are named as GBB-bPLA2, GBB-MP and GBB-TLE, respectively. Results from inductively coupled plasma atomic emission spectrometry(ICP-AES) indicate that GBB-bPLA2 and GBB-MP are Ca2+ containing proteins with the calcium stoichiometry of 1∶1([Ca2+]/[protein]), and GBB-TLE is a Zn2+ protein with a molar ratio of 2∶1([Zn2+]/[protein]). Ca2+ is critical for the enzymatic activities of GBB-bPLA2 and GBB-MP. Ca2+ ion induced 2.0 nm blue-shift and 1.6 nm blue-shift for the intrinsic fluorescences of GBB-bPLA2 and GBB-MP, respectively. The existence of Ca2+ also increased the maximum emission fluorescence intensities of GBB-bPLA2 and GBB-MP by 14.0% and 11.0%, respectively. Ca2+ could raise the denaturing temperature of GBB-bPLA2 and GBB-MP by 1.5 and 2.0 ℃, which resulted in higher thermal stabilities for them. Zn2+ ion could enhance the fluorescence intensity of GBB-TLE by 4.3%, however it did not show apparent influence on its esterase hydrolytic activity. Zn2+ only showed slight effect on the emission fluorescence wavelength and thermal stability of GBB-TLE. The existence of metal ions apparently affects the structure-related thermal stabilities of the metal-containing proteins of snake venom to different extents, meanwhile, the metal ions exhibit diverse influences on their enzymatic activities.

Key words: Snake venom protein; Metal ion; Activity

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