Li H, Otvos J D
Department of Food Science, North Carolina State University, Raleigh 27695, USA.
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。
