Reaction of 111Cd7-metallothionein with EDTA. A reappraisal.
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 beta-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 microM 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-alpha- and Cd3-beta-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-beta 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.
Gan
T
T
Department of Chemistry, University of Wisconsin-Milwaukee 53201.
Munoz
A
A
Shaw
C F
CF
Petering
D H
DH
D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism*; D002850:Chromatography, Gel; D004492:Edetic Acid / Q000378:metabolism*; D007554:Isotopes; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008668:Metallothionein / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D015032:Zinc
D007554:Isotopes; C004203:cadmium-binding protein; C021267:zinc thionein; D002104:Cadmium; D008668:Metallothionein; D004492:Edetic Acid; D015032:Zinc