Assay of copper transfer and binding to P1B–ATPases

Abstract

P1B-type ATPases transport transition metals across biological membranes. The chemical characteristics of these substrates, as well as, physiological requirements have contributed to the evolution of high metal binding affinities (fM) in these enzymes. Metal binding determinations are consequently facilitated by the stable metal–protein interaction, while affinity measurements require careful analysis of metal levels. In the cell, transition metals are associated with chaperone proteins. Metals reach the ATPase transport sites following specifi c protein–protein interactions and ligand exchange enabling the metal transfer from the chaperone to the transporter. Here, we describe methods for analyzing the binding of Cu+ to Cu+-ATPases, as well as the approach to monitor Cu+ transfer from soluble Cu+-chaperones donors to and from membrane Cu+-ATPases.