稀土离子Tb<SUP>3+</SUP>与拟南芥钙调素相互作用的荧光光谱及分析应用

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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(221 nm)或敏化激发(280 nm)并测量545 nm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(221 nm) 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c_Tb³⁺/c_CaM＜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c_Tb³⁺/c_CaM≤2时，荧光强度增加较弱;当2＜c_Tb³⁺/c_CaM＜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c_Tb³⁺/c_CaM=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，稳定性较好等优点。

关键词：铽离子;荧光光谱;钙调素;拟南芥

Abstract：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 Ⅳ 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 Ⅱ 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.

Key words：Tb³⁺;Fluorescence spectrum;Calmodulin;Arabidopsis thaliana

收稿日期: 2003-09-26 修订日期: 2004-02-20

中图分类号:

O657.3

通讯作者: 肖凤娟

引用本文:

肖凤娟¹，刘德龙²，白娟³，孙大业³ . 稀土离子Tb³⁺与拟南芥钙调素相互作用的荧光光谱及分析应用 [J]. 光谱学与光谱分析, 2004, 24(08): 984-987.
XIAO Feng-juan¹, LIU De-long², BAI Juan³, SUN Da-ye³ . Fluorescence of Tb³⁺-Calmodulin Complex and Its Analytical Application . SPECTROSCOPY AND SPECTRAL ANALYSIS, 2004, 24(08): 984-987.

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http://www.gpxygpfx.com/CN/Y2004/V24/I08/984