【摘要】 W 7-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W 7-agarose与植物胞外钙调素的相互结合反应。W 7-agarose是一种将W 7-共价连接到颗粒型agarose(琼脂糖)的粒子。W 7-agarose颗粒较大且容易沉淀,静置5m in后,溶液中的荧光强度完全由游离的CaM产生。在溶液中加入W 7-agarose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4.9×105。由此进一步计算了W 7-agarose对胞外钙调素的拮抗率,在拮抗剂W 7-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W 7-agarose与CaM的结合作用。更多还原

【Abstract】 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 W7-（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{_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 4.9×10⁵.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.更多还原