植物胞外钙调素与拮抗剂W7-Agarose结合作用的荧光光谱法研究  

Study on the Interaction of Plant Extracellular Calmodulin and Antagonist W7-agarose by Fluorescence Method

作　　者：刘德龙[1] 王晓静[1] 白娟[2] 孙大业[2] 

机构地区：[1]河北师范大学化学学院,石家庄050016 [2]河北师范大学生命科学学院,石家庄050016

出　　处：《分析化学》2006年第2期169-172,共4页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.20475013);国家科技部973资助项目(No.G19990117)

摘　　要：W7-agarose是常用的细胞外CaM功能的拮抗剂，本实验采用荧光光谱法研究了水溶液中钙调素拮抗剂W7-agarose与植物胞外钙调素的相互结合反应。W7-agarose是一种将W7-共价连接到颗粒型agamse（琼脂糖）的粒子。W7-agarose颗粒较大且容易沉淀，静置5min后，溶液中的荧光强度完全由游离的CaM产生。在溶液中加入W7-agamse后，溶液中一部分CaM与其结合后沉降至荧光比色皿底部，导致溶液中CaM的荧光强度下降。由此可以确定溶液中游离CaM的浓度。根据公式lg{[Q]t（F0-F）／F0}=nlg{[Q]tF/F0}＋lgnK[B]t，从而计算出配位体系的结合常数和配比。研究表明：二者以摩尔比1：1结合，其平衡常数为4．9×10^5。由此进一步计算了W7-agarose对胞外钙调素的拮抗率，在拮抗剂W7-agarose浓度达到15～20μmol／L时，拮抗率可达到90％以上，与文献报道的生物学体内实验结果一致，从分子水平上解释了W7-agarose与CaM的结合作用。W7-agarose is the antagonist of plant extracellular calmodulin in common use. The binding reaction between calmodulin （CaM） antagonist W7-agarose and plant extracellular calmodulin in aqueous solution was studied by fluorescence method. W7-agarose, in which W7- covalently linked to agarose beads, is even larger than a cell, and it also deposited quickly （after 5 minutes, it can deposit completely）. When adding WT- agarose to CaM solution, the bound CaM molecules deposited to the bottom and the free CaM is still in the solution. The fluorescence intensity in the liquor is completely from the free CaM molecule. Thereby, the concentration can be ascertained of the free CaM in liquor. Based on the equation lg{ [ Q ]t （F0 - F）/F0} = nlg{ [Q]tF/F0} ＋ lg nK[ B]t,the molar ratio and the equilibrium constant K can be clculated. The results indicate that W7-agarose binds to plant extracellular calmodulin with a molar ratio of 1.1, and the equilibrium constant K is 4.9 × 10^5. Furthermore, the antagonistic percentage of CaM can be calculated. When the concentration of antagonist W7-agarose reaches 15 -20 μmol/L, the antagonistic percentage exceeds 90%, which is consistent with the in vivo experiments in literatures. The experiments made a quantitative explanation of the binding reaction between calmodulin antagonist W7-agarose and plant extracellular calmodulin at molecule level.

关 键 词：钙调素 W7-agarose拮抗剂 结合常数 荧光 

分 类 号：Q942[生物学—植物学]