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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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

Received: 2003-09-26 Accepted: 2004-02-20

ZTFLH:

O657.3

Corresponding Authors: XIAO Feng-juan

Cite this article:

XIAO Feng-juan,LIU De-long,BAI Juan, et al. Fluorescence of Tb³⁺-Calmodulin Complex and Its Analytical Application [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2004, 24(08): 984-987.

URL:

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