Otvos J D, Armitage I M
Proc Natl Acad Sci U S A. 1980 Dec;77(12):7094-8. doi: 10.1073/pnas.77.12.7094.
Cadmium-113 nuclear magnetic resonance ((113)Cd NMR) has been used to determine the structures of the multiple cadmium binding sites in the two major isoproteins of rabbit liver metallothionein. The isotopically (113)Cd-labeled metallothionein used in these studies was isolated from the livers of rabbits that had been subjected to repeated injections of (113)CdCl(2). The native protein isolated from these livers contains an appreciable amount of Zn in addition to Cd, ranging from 2-3 mol per mol of protein out of a total metal content of 7 mol per mol of protein. The (113)Cd NMR spectrum of Cd, Zn-containing metallothionein is quite complex, reflecting the fact that the native protein is a heterogeneous mixture of species containing different relative amounts of Zn and Cd. Replacement of the native Zn with (113)Cd in vitro gave a protein whose (113)Cd NMR spectrum was much simpler, containing eight distinct multiplets with chemical shifts ranging from 611-670 ppm. The origin of the multiplet structures has been shown to be (113)Cd-(113)Cd scalar coupling arising from two-bond interactions between (113)Cd ions linked to one another by bridging cysteine thiolate ligands. The size and structures of the metal clusters in the protein were determined by the application of selective homonuclear (113)Cd decoupling techniques. Analysis of these data showed that rabbit liver metallothionein contains two separate metal clusters, one containing four Cd(2+) ions and the other containing three. These two clusters, whose structures are the same in both isoproteins, have been designated "cluster A" and "cluster B," respectively. Structures for the clusters are proposed that account for the (113)Cd spin coupling data and the participation of all 20 of the cysteine residues in metal ligation, 11 in cluster A and 9 in cluster B. The appearance in the spectrum of eight multiplets rather than the seven that would be expected on the basis of the number of metal binding sites in the protein is an indication of some residual heterogeneity in the (113)Cd-labeled metallothionein sample. The origin of this heterogeneity is suggested to be the presence of a protein species that lacks metal ions at its cluster B binding sites.
镉 - 113核磁共振（(113)Cd NMR）已被用于确定兔肝金属硫蛋白两种主要同工蛋白中多个镉结合位点的结构。这些研究中使用的同位素(113)Cd标记的金属硫蛋白是从经反复注射(113)CdCl₂的兔肝脏中分离得到的。从这些肝脏中分离出的天然蛋白除镉外还含有相当数量的锌，在每摩尔蛋白总金属含量为7摩尔的情况下，每摩尔蛋白含2 - 3摩尔锌。含镉、锌的金属硫蛋白的(113)Cd NMR谱相当复杂，这反映出天然蛋白是含有不同相对量锌和镉的物种的异质混合物。在体外将天然锌用(113)Cd替代后得到一种蛋白，其(113)Cd NMR谱要简单得多，包含八个不同的多重峰，化学位移范围为611 - 670 ppm。已证明多重峰结构的起源是由通过桥连半胱氨酸硫醇盐配体相互连接的(113)Cd离子之间的双键相互作用产生的(113)Cd-(113)Cd标量耦合。通过应用选择性同核(113)Cd去耦技术确定了蛋白中金属簇的大小和结构。对这些数据的分析表明，兔肝金属硫蛋白含有两个独立的金属簇，一个含有四个Cd²⁺离子，另一个含有三个。这两个簇在两种同工蛋白中的结构相同，分别被命名为“簇A”和“簇B”。提出了簇的结构，该结构解释了(113)Cd自旋耦合数据以及所有20个半胱氨酸残基参与金属配位的情况，其中11个在簇A中，9个在簇B中。谱中出现八个多重峰而非根据蛋白中金属结合位点数量预期的七个多重峰，表明(113)Cd标记的金属硫蛋白样品存在一些残留的异质性。这种异质性的起源被认为是存在一种在其簇B结合位点缺乏金属离子的蛋白物种。
