摘要
钙调素 (CaM)是一种普遍存在的钙受体蛋白 ,它调节了许多细胞生物学功能。钙调素分子有 4个金属结合位点 ,而植物钙调素Ⅰ ,Ⅱ ,Ⅲ位点不含酪氨酸残基 ,只有第 4位点含一个酪氨酸残基。与动物体不同的是 ,植物能表达多种功能不同的钙调素亚型。文章利用Tb3 +荧光探针 ,采用直接激发 ( 2 2 1nm)或敏化激发 ( 2 80nm)并测量 5 4 5nm处的荧光发射强度 ,研究拟南芥CaM与Tb3 +的结合作用及分析应用。直接激发 ( 2 2 1nm)Tb3 + CaM络合物时 ,Tb3 +的发光显著增强 ,这归因于CaM中配位基取代了Tb3 +的配位水分子 ,从而导致荧光速率常数增加。直接激发滴定曲线中 ,当cTb3+/cCaM <4时 ,荧光强度呈不断上升的直线 ,之后出现平台区 ,这与CaM只结合 4个钙离子的结论一致。而间接激发滴定曲线近似为S型 ,且荧光强度较弱 ;其中当cTb3+/cCaM≤ 2时 ,荧光强度增加较弱 ;当 2 <cTb3+/cCaM<4时 ,荧光强度增加较大 ,这是由于与Tb3 +配位的CaM的弱结合位点中的酪氨酸残基发生显著的能量传递之故。进一步根据两类滴定曲线均在cTb3+/cCaM=4时呈现明显转折点的事实 ,建立一种测定钙调素浓度的方法。此方法估测CaM浓度具有灵敏度 ,准确度较高 。
Calmodulin (CaM) is a ubiquitous Ca2+-binding protein of eukaryotes, and regulates a broad spectrum of fundamental cellular processes. CaM harbors four binding domains, among which domains I, II and III contain no tyrosine, only domain W has one tyrosine for plant species. In contrast to mammals, plants express numerous CaM isoforms that exhibit differential activation or inhibition of CaM dependent enzymes in vitro. In the present study, the isoform. U of Arabidopsis thaliana CaM was used to test the binding properties of metal ion to CaM by Tb3+ fluorescence. The increase in fluorescence was monitored at 545 nm as a function of the number of Tb3+ bound to CaM using direct (221 nm) and indirect (280 nm) excitation. Upon direct excitation of Tb3+-CaM, the fluorescence of Tb3+ increased markedly, and one of the pathways of energy dissipation of the excited state of Tb3+ was energy transfer to the vibrational levels of water molecules in the hydration sphere around the Tb3+ ion. When waters of hydration were removed as a result of Tb3+ binding to CaM, an increase in rate constants of luminescence was observed. The titration curve with direct excitation increased up to 4 mol of Tb3+/mol of CaM before the onset of a plateau, in agreement with the expected maximum of four binding sites. Using indirect excitation at 280 nm, the resultant titration curve was sigmoid, albeit with less fluorescence intensity, also reached a maximum at a ratio of 4 mol of Tb3+/mol of CaM, in which the first phase exhibits an end at 2:1, and in this phase there was only a small increase in Tb3+ fluorescence. The fact that only the second pair of added Tb3+ shows a large enhancement in Tb3+ fluorescence suggests that it is Tb3+ bound to the low affinity sites that can accept energy from tyrosine group. The turning points of fluorescence titration curves were used to estimate the concentrations of CaM on the base of c(Tb3+) = 4c(CaM).
出处
《光谱学与光谱分析》
SCIE
EI
CAS
CSCD
北大核心
2004年第8期984-987,共4页
Spectroscopy and Spectral Analysis
基金
国家自然科学基金重点项目 (39730 2 30 )
河北省博士基金项目资助
