银离子和铜离子与锌金属硫蛋白和镉金属硫蛋白发生金属交换反应的高效液相色谱表征
HPLC characterization of Ag+ and Cu+ metal exchange reactions with Zn- and Cd-metallothioneins.
作者信息
Li H, Otvos J D
机构信息
Department of Food Science, North Carolina State University, Raleigh 27695, USA.
出版信息
Biochemistry. 1996 Nov 5;35(44):13937-45. doi: 10.1021/bi961402f.
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.
开发了阴离子交换高效液相色谱法来研究锌金属硫蛋白（ZnMT）和镉金属硫蛋白（CdMT）与银离子（Ag⁺）和铜离子（Cu⁺）的金属交换反应。这些反应的动力学揭示了在Ag⁺或Cu⁺最初快速取代Zn₇MT或Cd₇MT中的金属之后，存在一个缓慢的蛋白质间金属重新分布过程。在螯合树脂存在下，Ag⁺与Zn₇MT反应的动力学有利产物包括Zn₄Ag₆MT和Ag₁₂MT。随后，Zn₇MT和Ag₁₂MT之间的缓慢反应产生了热力学有利产物Zn₄Ag₆MT。在用Ag⁺滴定Cd₇MT以及用Cu⁺滴定Zn₇MT时观察到了类似的行为。发现使用螯合树脂去除未结合的金属离子会影响Zn₇MT和Cd₇MT与Ag⁺的反应。在没有螯合树脂的情况下向Zn₇MT中添加Ag⁺时，除了Zn₄Ag₆MT和Ag₁₂MT之外，还会形成动力学有利产物Ag₁₈MT。随后，发现Ag₁₈MT通过缓慢的蛋白质间金属交换反应将Ag⁺转移给Zn₇MT和Zn₄Ag₆MT。螯合树脂通过限制Ag₁₈MT的形成和/或通过结合带正电荷的Ag₁₈MT物种并将其从溶液中去除来影响滴定反应。在没有螯合树脂的情况下形成Ag₁₈MT解释了为什么需要超过12当量的Ag⁺才能从Zn₇MT和Cd₇MT中取代所有的Zn²⁺或Cd²⁺。本研究中开发的高效液相色谱法能够制备出在β结构域含有Ag⁺且在α结构域含有Cd²⁺或Zn²⁺的金属杂化金属硫蛋白物种的均一制剂。
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