Área de Química Orgánica, Faculty of Experimental Sciences, University of Almería, La Cañada de San Urbano, 04120 Almería, Spain.
Biochimie. 2012 Feb;94(2):541-50. doi: 10.1016/j.biochi.2011.09.003. Epub 2011 Sep 17.
The binding properties of two electroactive glutathione-ferrocene conjugates that consist in glutathione attached to one or both of the cyclopentadienyl rings of ferrocene (GSFc and GSFcSG), to Schistosoma japonica glutathione S-transferase (SjGST) were studied by spectroscopy fluorescence, isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Such ferrocene conjugates resulted to be competitive inhibitors of glutathione S-transferase with an increased binding affinity relative to the natural substrate glutathione (GSH). We found that the conjugate having two glutathione units (GSFcSG) exhibits an affinity for SjGST approximately two orders of magnitude higher than GSH. Furthermore, it shows negative cooperativity with the affinity for the second binding site two orders of magnitude lower than that for the first one. We propose that the reason for such negative cooperativity is steric since, i) the obtained thermodynamic parameters do not indicate profound conformational changes upon GSFcSG binding and ii) docking studies have shown that, when bound, part of the first bound ligand invades the second site due to its large size. In addition, voltammetric measurements show a strong decrease of the peak current upon binding of ferrocene-glutathione conjugates to SjGST and provide very similar K values than those obtained by ITC. Moreover, the sensing ability, expressed by the sensitivity parameter shows that GSFcSG is much more sensitive than GSFc, for the detection of SjGST.
两种电化学活性谷胱甘肽-二茂铁缀合物（GSFc 和 GSFcSG，分别由一个或两个环戊二烯基环上的谷胱甘肽连接到二茂铁上）与日本血吸虫谷胱甘肽 S-转移酶（SjGST）的结合特性通过光谱荧光、等温滴定微量热法（ITC）和差示脉冲伏安法（DPV）进行了研究。这些二茂铁缀合物是谷胱甘肽 S-转移酶的竞争性抑制剂，与天然底物谷胱甘肽（GSH）相比，其结合亲和力增加。我们发现，具有两个谷胱甘肽单元的缀合物（GSFcSG）对 SjGST 的亲和力大约比 GSH 高两个数量级。此外，它与第二个结合位点的亲和力呈负协同性，比第一个结合位点的亲和力低两个数量级。我们提出，这种负协同性的原因是空间位阻，因为：i）获得的热力学参数表明在 GSFcSG 结合时没有明显的构象变化，ii）对接研究表明，当结合时，由于其较大的尺寸，第一个结合配体的一部分会侵入第二个结合位点。此外，伏安测量表明，当 ferrocene-glutathione 缀合物与 SjGST 结合时，峰电流会强烈下降，并且提供的 K 值与 ITC 获得的值非常相似。此外，灵敏度参数表示 GSFcSG 比 GSFc 对 SjGST 的检测更灵敏。
