Spectroscopic and thermodynamic studies on the binding of gadolinium(III) to human serum transferrin.
Spectroscopic and thermodynamic studies on the binding of gadolinium(III) to human serum transferrin.
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DOI：
10.1021/bi00403a033
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发表时间：
1988-02
期刊：
影响因子：
2.9
通讯作者：
O. Zak;P. Aisen
中科院分区：
文献类型：
--
作者：
O. Zak;P. Aisen
各种各样的热力学，动力学和光谱学研究已经证明了运铁蛋白的两个金属结合位点之间的差异。在本研究中，我们进一步评估了这些差异，相对于钆的结合，通过紫外差分光光度法，电子顺磁共振（EPR）滴定，EPR差光谱结合尿素凝胶电泳，平衡透析。这些研究的组合确定，只有一个网站的蛋白质结合Gd（III）足够牢固的特点。为了揭示这两个网站接受Gd（III），我们利用单铁转铁蛋白优先加载Fe（III）在任一网站的EPR光谱研究。由于信号的重叠，需要差分光谱来区分由Fe（III）和Gd（III）特异性地与蛋白质复合而产生的共振。当铁结合到C-末端位点，使N-末端位点自由结合钆时，差异光谱显示没有特异性结合的证据。然而，当铁结合到N-末端位点时，差谱显示在g' = 4.1处的共振线，指示特异性结合，因此暗示Gd（III）结合中的C-末端位点。在pH7.4和环境pCO 2下，Gd（III）与转铁蛋白该位点结合的有效稳定常数为6.8 × 10（6）M-1。在生理pCO 2，非结合碳酸配合物的Gd（III）的形成排除了转铁蛋白在体内的镧系元素的运输中的重要作用。
A wide variety of thermodynamic, kinetic, and spectroscopic studies have demonstrated differences between the two metal-binding sites of transferrin. In the present investigation, we have further assessed these differences with respect to the binding of gadolinium, evaluated by UV difference spectrophotometry, electron paramagnetic resonance (EPR) titration, EPR difference spectroscopy in conjunction with urea gel electrophoresis, and equilibrium dialysis. Combinations of these studies establish that only one site of the protein binds Gd(III) sufficiently firmly to be characterized. In order to reveal which of the two sites accepts Gd(III), we made use of monoferric transferrins preferentially loaded with Fe(III) at either site in EPR spectroscopic studies. Because of the overlap of signals, difference spectroscopy was required to distinguish resonances arising from Fe(III) and Gd(III) specifically complexed to the protein. When iron is bound to the C-terminal site, leaving the N-terminal site free for binding of gadolinium, the difference spectrum shows no evidence of specific binding. However, when iron is bound to the N-terminal site, the difference spectrum shows a resonance line at g' = 4.1 indicative of specific binding, thus implicating the C-terminal site in the binding of Gd(III). The effective stability constant for the binding of Gd(III) to this site of transferrin at pH 7.4 and ambient pCO2 is 6.8 X 10(6) M-1. At physiological pCO2, the formation of nonbinding carbonato complexes of Gd(III) precludes a substantial role for transferrin in the transport of the lanthanide in vivo.