Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol
Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol
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DOI：
10.1016/j.ab.2019.03.007
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发表时间：
2019-08-15
影响因子：
2.9
通讯作者：
Meiering, Elizabeth M.
中科院分区：
文献类型：
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作者：
Doyle, Colleen M.;Naser, Dalia;Meiering, Elizabeth M.
在许多天然蛋白质中都观察到了结合金属，它们在决定蛋白质折叠、稳定性和功能方面发挥着不同的作用。由于结合金属对蛋白质的生物物理和生化特性具有广泛的影响，因此拥有准确且简便的方法来测定蛋白质的金属含量是很有价值的。在这里，我们描述了一种使用 4-(2-吡啶偶氮)间苯二酚 (PAR) 同时定量溶液中两种金属离子的优化方法。该测定法可用于对人铜、锌超氧化物歧化酶 (SOD1) 中的 Cu2+ 和 Zn2+ 离子进行定量；然而，该方法是通用的，可以应用于金属离子的各种组合。该测定的优点是快速且廉价，需要很少的样品和制备，并且数据分析简单。我们表明，光谱分解软件可以高精度地准确解析 Cu2+ 和 Zn2+ 的吸收带。使用 PAR 测定，我们确定与疾病相关的 SOD1 突变体中金属结合发生改变，结果与 ICP-AES 测定的结果相当。此外，我们还重点介绍了使用分光光度螯合剂（例如 PAR）进行蛋白质金属分析的关键问题。
Bound metals are observed in a great many natural proteins, where they perform diverse roles in determining protein folding, stability and function. Due to the broad impact of bound metals on biophysical and biochemical properties of proteins, it is valuable to have accurate and facile methods for determining the metal content of proteins. Here we describe an optimized methodology using 4-(2-pyridylazo)resorcinol (PAR) to simultaneously quantify two metal ions in solution. The assay is demonstrated for quantification of Cu2+ and Zn2+ ions in human Cu, Zn superoxide dismutases (SOD1s); however, the method is general and can be applied to various combinations of metal ions. Advantages of the assay are that it is rapid and inexpensive, requires little sample and preparation, and has simple data analysis. We show that spectral decomposition software can accurately resolve the absorption bands of Cu2+ and Zn2+ with high accuracy and precision. Using the PAR assay, we determined that metal binding is altered in disease-associated mutants of SOD1, with comparable results to those determined by ICP-AES. In addition, we highlight key issues for using spectrophotometric chelators such as PAR for metal analysis of proteins.