Deitrich Christian L, Raab Andrea, Pioselli Barbara, Thomas-Oates Jane E, Feldmann Jörg
College of Physical Science, Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen, UK AB24 3UE.
Anal Chem. 2007 Nov 1;79(21):8381-90. doi: 10.1021/ac071397t. Epub 2007 Sep 25.
The development of methods to analyze accurately and precisely individual metalloproteins is of increasing importance. Here we describe for the first time the chemical preparation and characterization of an isotopically enriched metalloenzyme containing two different metal isotopes. Its evaluation as a standard in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is carefully evaluated. Our model enzyme bovine superoxide dismutase (SOD) contains both Cu and Zn and is remarkably stable at high temperatures and even under denaturing conditions. The enzyme's metal cofactors were removed under a range of different conditions and replaced with isotopically enriched 65Cu and 68Zn. Depending on the conditions, various isotopic ratios differing from the natural Cu and Zn abundances were obtained for the reconstituted enzyme. Both the wild type and isotopically enriched enzyme had the same migration pattern on native 1D-PAGE. Using an enzyme activity test, we showed that the incorporated 65Cu was bound to the right SOD-binding site, since the measured activity correlated directly with the amount of Cu incorporated. Mixing the native and the isotopically enriched enzyme standard with free enriched 65Cu and 68Zn or a metal chelator did not result in any exchange or loss of the metals from the enzyme at neutral pH. This verifies the stability of the enzyme metal center under the chosen conditions. The isotopically enriched enzyme standard was spiked into a wild type SOD solution to evaluate its use for species-specific isotope dilution experiments. To our knowledge, this is the first report of the chemical preparation of a metalloenzyme containing two different isotopically enriched metals. We provide evidence that the incorporated isotopically enriched metals are bound to the right binding site of SOD using an specific enzymatic activity assay.
准确且精确地分析单个金属蛋白的方法的发展变得越来越重要。在此，我们首次描述了一种含有两种不同金属同位素的同位素富集金属酶的化学制备及表征。仔细评估了其作为通过与电感耦合等离子体质谱联用的高效液相色谱进行物种特异性同位素稀释分析的标准物的性能。我们的模型酶牛超氧化物歧化酶（SOD）同时含有铜和锌，并且在高温甚至变性条件下都非常稳定。在一系列不同条件下去除该酶的金属辅因子，并用同位素富集的⁶⁵Cu和⁶⁸Zn进行替换。根据条件不同，重构后的酶获得了与天然铜和锌丰度不同的各种同位素比率。野生型酶和同位素富集酶在天然一维聚丙烯酰胺凝胶电泳上具有相同的迁移模式。通过酶活性测试，我们表明掺入的⁶⁵Cu与正确的SOD结合位点结合，因为测得的活性与掺入的铜量直接相关。在中性pH条件下，将天然酶和同位素富集酶标准物与游离的富集⁶⁵Cu和⁶⁸Zn或金属螯合剂混合，不会导致酶中金属的任何交换或损失。这验证了在所选条件下酶金属中心的稳定性。将同位素富集的酶标准物加入野生型SOD溶液中，以评估其在物种特异性同位素稀释实验中的用途。据我们所知，这是关于含有两种不同同位素富集金属的金属酶化学制备的首次报道。我们通过特异性酶活性测定提供证据表明，掺入的同位素富集金属与SOD的正确结合位点结合。
