Development of affinity bead-based <i>in vitro</i> metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione
Development of affinity bead-based <i>in vitro</i> metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione
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基于亲和珠的<i>体外</i>金属配体结合测定揭示了除谷胱甘肽之外富含硫醇的小肽植物螯合素的主要镉亲和力
DOI：
10.1093/mtomcs/mfab068
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发表时间：
2021
期刊：
影响因子：
3.4
通讯作者：
Kiyono Masako
中科院分区：
文献类型：
--
作者：
Uraguchi Shimpei;Nagai Kenichiro;Naruse Fumii;Otsuka Yuto;Ohshiro Yuka;Nakamura Ryosuke;Takanezawa Yasukazu;Kiyono Masako
为了更好地理解金属-配体相互作用及其在细胞中的功能，我们开发了一种简单、灵敏、高通量的方法，通过结合配体附着的亲和珠和电感耦合等离子体发射光谱法（ICP-OES）来定量生理条件下的配体-金属（类）结合亲和力。谷胱甘肽（GSH）和两种植物螯合素（PC 2和PC 3，具有不同数量的游离巯基的小肽）作为模型配体，并连接到亲水性珠。该测定法的原理类似于生物化学中蛋白质的亲和纯化：通过旋转柱分离与珠子上的配体结合的金属和缓冲液中的其余金属，并通过ICP-OES进行定量。使用GSH附着的珠和各种金属（类）的结合测定表明金属-GSH相互作用的不同亲和力，与Irving-Williams系列的顺序和报道的稳定常数一致。使用PC 2或PC 3连接珠的结合试验表明PC和Ni（II），Cu（II），Zn（II），Cd（II）和As（III）之间的正结合，根据PC 2和PC 3中的硫醇的数量。然后，我们进行了竞争试验，使用镉（II），锰（II），铁（II），铜（II），和锌（II），结果表明，更好的结合亲和力的PC 2与镉（II）比与必需金属。另一个竞争试验，使用PC 2和GSH表明一个强大的PC和Cd（II）之间的结合亲和力相比，GSH和Cd（II）。这些结果表明，在体外PC-Cd复合物的形成占主导地位，支持PC的解毒镉在体内的生理意义。我们还讨论了潜在的应用的测定。
For a better understanding of metal–ligand interaction and its function in cells, we developed an easy, sensitive, and high-throughput method to quantify ligand–metal(loid) binding affinity under physiological conditions by combining ligand-attached affinity beads and inductively coupled plasma-optical emission spectrometry (ICP-OES). Glutathione (GSH) and two phytochelatins (PC2 and PC3, small peptides with different numbers of free thiols) were employed as model ligands and attached to hydrophilic beads. The principle of the assay resembles that of affinity purification of proteins in biochemistry: metals binding to the ligand on the beads and the rest in the buffer are separated by a spin column and quantified by ICP-OES. The binding assay using the GSH-attached beads and various metal(loid)s suggested the different affinity of the metal–GSH interactions, in accordance with the order of the Irving–Williams series and the reported stability constants. The binding assay using PC2 or PC3-attached beads suggested positive binding between PCs and Ni(II), Cu(II), Zn(II), Cd(II), and As(III) in accordance with the number of thiols in PC2 and PC3. We then conducted the competition assay using Cd(II), Mn(II), Fe(II), Cu(II), and Zn(II), and the results suggested a better binding affinity of PC2 with Cd(II) than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and Cd(II) compared to GSH and Cd(II). These results suggested the dominance of PC-Cd complex formationin vitro, supporting the physiological importance of PCs for the detoxification of cadmiumin vivo. We also discuss the potential application of the assay.