Characterization of Cd, Zn-thionein (metallothionein) isolated from rat and chicken liver.
Characterization of Cd, Zn-thionein (metallothionein) isolated from rat and chicken liver.
复制标题
从大鼠和鸡肝中分离的镉、锌硫蛋白（金属硫蛋白）的表征。
DOI：
10.1111/j.1432-1033.1973.tb03111.x
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发表时间：
1973
期刊：
影响因子：
--
通讯作者：
G. Jung
中科院分区：
文献类型：
--
作者：
U. Weser;H. Rupp;F. Donay;F. Linnemann;W. Voelter;W. Voetsch;G. Jung
从镉预处理的大鼠和鸡的纯金属硫蛋白的制备进行使用短时间的处理，氯仿/乙醇，然后通过离子交换和凝胶色谱。每12000 g蛋白质含有7 g原子金属离子。锌镉比为1：2.4 ± 0.1。Cd，Zn硫蛋白在Sephadex G-25，G-50和G-75凝胶过滤，聚丙烯酰胺盘状凝胶电泳和分析超滤中保持均一。高含量的半胱氨酸残基（约总残基的三分之一）与分离自人或马组织的金属硫蛋白的半胱氨酸含量一致。镉锌硫蛋白具有较好的温度稳定性。通过紫外光谱、圆二色性（CD）和X射线光电子能谱（XPS）等测试手段，对样品的理化性质进行了研究。天然蛋白质的毫摩尔吸收系数在250 nm处为λ 250= 80.6 mM−1· cm−1。实际上不存在芳香族氨基酸。通过使用HCl的金属置换制备的脱辅基蛋白的Δ 250值为Δ 250= 13.2mM-1· cm-1。当在pH 6.6下记录载脂蛋白的紫外光谱时，在255 nm处可检测到一个明显的峰（λ 250= 18.7 m M−1· cm−1）。采用二叔丁氧羰基-L-胱氨酸和叔丁氧羰基-L-半胱氨酸的聚乙二醇酯分别作为高分子量和水溶性半胱氨酸化合物的模型化合物，通过CD测量证实了分子间或分子内二硫化物形成的存在。
从CD数据可以得出结论，镉，锌-硫蛋白主要是作为一个无规卷曲肽。从紫外数据中获得的一些迹象表明，在天然蛋白质中的镉和锌的结合与BASIS-R部分。这些金属离子与半胱氨酸硫的独家协调的最终证明是成功的，使用圆二色性测量，特别是X射线光电子能谱。最后一种方法被证明是最方便的，用于确定Cd（Cd 3d ~（3/2）= 411.0 eV和Cd 3d ~（5/2）= 404.4 eV）、Zn（2 p ~（3/2）= 1021.0 eV）和S（2 p = 161.7 eV）的核心电子结合能。胱氨酸与Zn ~（2+）和Cd ~（2+）的配合物中S的结合能分别为163.0 eV和162.8 eV。
The preparation of pure metallothionein from Cd-pretreated rats and chicken was performed using short-time treatment with chloroform/ethanol followed by ion-exchange and gel chromatography. The protein contained 7 g-atoms metal ions per 12000 g protein. The Zn to Cd ratio was 1:2.4 ± 0.1. The Cd, Zn-thionein remained homogeneous during Sephadex G-25, G-50 and G-75 gel filtration, during polyacrylamide disc-gel electrophoresis and in the analytical ultracentrifuge. The high content of cysteine residues (approx. one-third of the total residues) was consistent with the cysteine content of metallothionein isolated from human or equine tissues. The Cd, Zn-thionein was of considerable temperature stability. The physicochemical properties were examined by ultraviolet spectroscopy, circular dichroism (CD) measurements and X-ray photoelectron spectroscopy. The millimolar absorption coefficient of the native protein was at 250 nm ɛ250= 80.6 mM−1· cm−1. Virtually no aromatic amino acids were present. The ɛ250 value of the apoprotein prepared by metal displacement using HCl was ɛ250= 13.2 mM−1· cm−1. When the ultraviolet spectrum of the apoprotein was recorded at pH 6.6 a distinct peak was detectable at 255 nm (ɛ250= 18.7 m M−1· cm−1). The existence of inter- or intramolecular disulphide formation was confirmed by CD measurements employing the polyethyleneglycol esters of di-tert-butyloxy-carbonyl-l-cystine and tert-butyloxycarbonyl-l-cysteine as model compounds for high-molecular-weight and water-soluble cysteine compounds, respectively.
From CD data it was concluded that Cd, Zn-thionein exists mainly as a random-coil peptide. Some indication of the binding of Cd and Zn in the native protein with ⊖S–R moieties was obtained from ultraviolet data. Final proof of the exclusive coordination of these metal ions with cysteine sulphur was successful using both circular dichroism measurements and especially X-ray photo-electron spectroscopy. The last method proved most convenient for determining the binding energies of the core electrons of Cd (Cd 3d3/2= 411.0 eV and Cd 3d5/2= 404.4 eV), Zn (2 p3/2= 1021.0 eV) and S (2p = 161.7 eV). The corresponding binding energies for S in the cystine complexes with Zn2+ and Cd2+ were 163.0 eV and 162.8 eV, respectively.