Reaction of ¹¹¹Cd₇-Metallothionein with EDTA

Abstract

The ligand substitution reaction of EDTA with Cd₇-metallothionein (Cd₇-MT) has been reinvestigated. NMR titration of the ¹¹¹Cd-protein with EDTA showed that the ligand interacts preferentially and cooperatively with Cd²⁺ ions in the β-domain cluster. NMR and ultrafiltration kinetic analysis of this reaction using 5.6 mM Cd²⁺ as ¹¹¹Cd₇-MT and 56 mM EDTA indicated that cadmium-EDTA formed less rapidly than ¹¹¹Cd peak intensity declined. Spectrophotometric and gel filtration studies of the reaction with 20 μM Cd²⁺ as Cd₇-MT with various concentrations of EDTA revealed biphasic kinetics with much larger rate constants than observed in the NMR experiments. The fraction of total ligand substitution occurring in each kinetic step varied with EDTA concentration. The EDTA concentration dependence of both kinetic steps was consistent with the initial formation of protein•EDTA adducts, followed by their breakdown into products. Kinetic measurements were also made for the reactions of the isolated Cd₄-α- and Cd₃-β-domains with EDTA. The Cd₄ domain reacted with EDTA with biphasic kinetics, in which one Cd²⁺ was removed rapidly with first-order kinetics, which were zero-order in EDTA. The other three reacted with kinetics like those for the slower step of the holoprotein. Cd₃-β reacted with EDTA like the faster rate process associated with the Cd₇-protein. The observed rate constants for the reaction of Cd₇-metallothionein with EDTA and the fraction of reaction in the faster rate process were sensitive to protein concentration. These results are consistent with the hypothesis that the monomer-dimer equilibrium of the protein controls its kinetic reactivity with EDTA.