Affiliation:
1. Department of Public Health, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
2. Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan
Abstract
Abstract
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 formation in vitro, supporting the physiological importance of PCs for the detoxification of cadmium in vivo. We also discuss the potential application of the assay.
Funder
Japan Society for the Promotion of Science
Publisher
Oxford University Press (OUP)
Subject
Metals and Alloys,Biochemistry,Biomaterials,Biophysics,Chemistry (miscellaneous)
Cited by
6 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献