Development of affinity beads-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione

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 thiol numbers) were employed as model ligands and attached to affinity beads. The principal 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 that the assay can detect the different affinity of the metal-ligand 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 nickel, copper, zinc, cadmium, and arsenite ions in a thiol-dependent manner. We then conducted the competition assay using cadmium, manganese, iron, copper, and zinc and the results suggested a better binding affinity of PC2 with cadmium than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and cadmium compared to GSH and cadmium. 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 further potential application of the assay.