物理化学学报 >> 1992, Vol. 8 >> Issue (05): 619-624.doi: 10.3866/PKU.WHXB19920509

研究论文 上一篇    下一篇

金属硫蛋白加质子常数及与Cd(II)络合常数测定

段震文; 王文清; 陈向阳; 王祥云; 茹炳根; 褚德萤; 林育   

  1. 北京大学技术物理系，北京 100871；北京大学生物系；北京大学化学系
  • 收稿日期:1991-05-22 修回日期:1991-11-04 发布日期:1992-10-15
  • 通讯作者: 王文清

Determination of Apo-Metallothionein's Protonation Constants and Complexing Constants With Cadmium Ion

Duan Zhen-Wen; Wang Wen-Qing; Chen Xiang-Yang; Wang Xiang-Yun; Ru Bing-Gen; Chu De-Ying; Lin Yu   

  1. Department of Technical Physics,Peking University, Beijing 100871; Department of Biology, Peking University; Department of Chemistry, Peking University
  • Received:1991-05-22 Revised:1991-11-04 Published:1992-10-15
  • Contact: Wang Wen-Qing

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摘要： 用pH计和Cd离子选择电极测定了金属硫蛋白的加质子常数及其与Cd(Ⅱ)的络合常数, 用改进的简化络合模型处理实验结果, 得到了去金属硫蛋白(apo MT)中6类不同的加质子基团的数目及其加质子常数。对Cd(Ⅱ)滴定数据的计算表明, MT中两个结构域——α和β对Cd(Ⅱ)的络合常数相差约1000倍。从热力学定量描述了MT中两个结构域结合金属离子选择优先顺序。

关键词: 金属硫蛋白, 加质子常数, Cd(II)络合常数

Abstract: Metallothionein (MT) is a protein of molecular weight about 6000 Dalton obtained from liver of rat, rabbit and silver carp in our laboratory. Mammal's MT consists of 61 amino acid residues including 20 cysteine residues which has high affinity to metal and it appears to coordinate metals in two distinct configurations. Ions of at least eighteen different metals were associated with the protein. Most metals exhibited saturation binding at 7 mol eq forming M_7-MT. These included Bi(Ⅲ), Cd(Ⅱ), Co(Ⅱ), Hg(Ⅱ), In(III), Ni(II), Pb(II), Sb(III), and Zn(II). Other metals including Os(III), Pd(II), Pt(IV), Re(V), Rh(III) and Tl(III) give a positive indication of binding but their stoichiometries remained unclear. Ag(I) and Cu(I) bound in clusters as M_(12)-MT.
In this paper, rabbit liver MT which combined with 5 Cd(II) and 2 Zn(II) ion per protein was obtained by gel filtration and ion exchange after homogenization, centrifugation and deposition with ethanol from rabbits received cadmium chloride by subcutaneously injection. In 0.01 mol·L~(-1) perchloric acid medium, the metal ions released from MT and apo-MT were obtained by gel filtration on a Sephadex G-25 column, the titration with sodium hydroxide and cadmium nitrate solution were carried out by combined pH electrode and cadmium ion selective electrode under nitrogen at 298 K.
According to the sequences of amino acid from rabbit liver MT-2, the binding sites were divide into six types. By using a simplified complexing model, six types of protonation group and two types of metal complexing region (α- and β- domain) were obtained. The results were listed as follows.
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Key words: Metallothionein, Protonation constants, Complexing constants of Cd(II) ion