Acta Phys. -Chim. Sin. ›› 1992, Vol. 8 ›› Issue (05): 619-624.doi: 10.3866/PKU.WHXB19920509

• ARTICLE • Previous Articles     Next Articles

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

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