Atanassova Anelia, Lam Robert, Zamble Deborah B
Department of Chemistry, University of Toronto, Lash Miller Chemical Laboratories, 80 St. George St., Toronto, Ont., Canada M5S 3H6.
Anal Biochem. 2004 Dec 1;335(1):103-11. doi: 10.1016/j.ab.2004.08.013.
Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed.
过渡金属是细胞蛋白质的常见组成部分，对金属蛋白的详细研究需要对结合的金属离子进行鉴定和定量。金属筛选也是目前通过蛋白质组学流程的众多未知和未分类蛋白质初步表征中的一个信息丰富的步骤。我们开发了一种高效液相色谱法，用于定量测定最常见的生物过渡金属：锰、铁、钴、镍、铜和锌。该方法准确且简单，可适用于自动化高通量研究。金属分析包括酸水解以将金属离子释放到溶液中，然后在混合珠离子交换柱上进行离子分离，并在用金属显色指示剂4-(2-吡啶偶氮)间苯二酚进行柱后衍生后进行吸光度检测。研究了蛋白质溶液常见成分的潜在干扰。分析了多种金属蛋白的金属含量，并将数据与电感耦合等离子体原子发射光谱法获得的数据进行了比较。该测定法的灵敏度允许检测0.1 - 0.8 nmol的金属，具体取决于金属种类。所需蛋白质的量由蛋白质的大小和结合金属的蛋白质部分决定。常规分析使用50微克，但对于许多蛋白质，10微克就足够了。讨论了该方法的优缺点及可能的应用。
