HPLC characterization of Ag+ and Cu+ metal exchange reactions with Zn- and Cd-metallothioneins.
Anion exchange HPLC methods were developed to study the metal exchange reactions of ZnMT and CdMT with Ag+ and Cu+. The kinetics of these reactions revealed the existence of a slow interprotein metal redistribution process that follows initial rapid displacement of metal from Zn7MT or Cd7MT by Ag+ or Cu+. Kinetically-favored products of the reaction of Ag+ with Zn7MT in the presence of Chelex resin include Zn4Ag6MT and Ag12MT. Subsequent slow reaction between Zn7MT and Ag12MT leads to the thermodynamically-favored product, Zn4Ag6MT. Analogous behavior was observed for Cd7MT titrated with Ag+ and Zn7MT with Cu+. The use of Chelex resin to remove unbound metal ions was found to influence the reactions of Zn7MT and Cd7MT with Ag+. Upon addition of Ag+ to Zn7MT in the absence of Chelex, a Ag18MT species forms as the kinetically-favored product in addition to Zn4Ag6MT and Ag12MT. Subsequently, Ag18MT was found to donate Ag+ to Zn7MT and Zn4Ag6MT via slow interprotein metal exchange reactions. Chelex resin influences the titration reaction either by limiting formation of Ag18MT and/or by binding the positively charged Ag18MT species and removing it from solution. The formation of Ag18MT in the absence of Chelex explains why more than 12 equiv of Ag+ is required to displace all of the Zn2+ or Cd2+ from Zn7MT and Cd7MT. The HPLC methods developed in this study allow homogeneous preparations to be made of metal-hybrid MT species containing Ag+ in the beta-domain and Cd2+ or Zn2+ in the alpha-domain.
Department of Food Science, North Carolina State University, Raleigh 27695, USA.
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D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007475:Ion Exchange Resins; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015394:Molecular Structure; D011137:Polystyrenes; D011145:Polyvinyls; D011485:Protein Binding; D011817:Rabbits; D012834:Silver / Q000737:chemistry / Q000378:metabolism*
D007475:Ion Exchange Resins; D011137:Polystyrenes; D011145:Polyvinyls; C004203:cadmium-binding protein; C021267:zinc thionein; D012834:Silver; D003300:Copper; C006960:chelex; D008668:Metallothionein