金属离子对蛇毒蛋白生物活性及结构效应的影响  

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

作　　者：刘淑清[1] 郭春梅[1,2] 徐跃飞[1] 孙明忠[2] 辛毅[2] 唐建武[3] 

机构地区：[1]大连医科大学生物化学与分子生物学教研室,大连116044 [2]大连医科大学基础医学院生物技术系 [3]大连医科大学病理学教研室,大连116044

出　　处：《高等学校化学学报》2009年第9期1773-1778,共6页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号:30572098;30772468);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1.6nm,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.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. Resuits from inductively coupled plasma atomic emission spectrometry （ICP-AES） indicate that GBB-bPLA2and GBB-MP are Ca^2＋ containing proteins with the calcium stoichiometry of 1：1 （ [ Ca^2＋ ]/[ protein ] ）, and GBBTLE is a Zn^2＋ protein with a molar ratio of 2：1 （ [ Zn^2＋ ]/[ protein ] ）. Ca^2＋ is critical for the enzymatic activities of GBB-bPLA2 and GBB-MP. Ca^2＋ 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 Ca^2＋ also increased the maximum emission fluorescence intensities of GBB-bPLA2 and GBB-MP by 14.0% and 11.0%, respectively. Ca^2＋ 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. Zn^2＋ ion could enhance the fluorescence intensity of GBB-TLE by 4.3%, however it did not show apparent influence on its esterase hydrolytic activity. Zn^2＋ 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.

关 键 词：蛇毒蛋白 金属离子 活性 

分 类 号：O629.7[理学—有机化学] O657[理学—化学]