Department of Chemistry, State University of New York at Potsdam, Potsdam, NY 13676, United States of America.
Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, NY 13699, United States of America.
J Inorg Biochem. 2021 Jul;220:111460. doi: 10.1016/j.jinorgbio.2021.111460. Epub 2021 Apr 15.
Iron is an essential nutrient for virtually all forms of life. Because of its redox properties and involvement in a wide range of biological processes, a number of qualitative and quantitative chemical tools have been developed to detect reduced (Fe) and oxidized (Fe) forms of iron in biomolecules. These types of measurements are not only important in detecting iron species in solution, but also in understanding iron distribution, accumulation, and role in physiological and pathological processes. Here, we use UV-vis spectrophotometry and three common chromogenic reagents, ferrozine, 2,2'-bipyridine, and 1,10-phenanthroline to detect and quantify the concentration of ferrous ions in aqueous solutions, owing to the unique absorption spectra, specific molar absorptivity, and characteristic colors of these Fe-chelator complexes. Our results show that the kinetics of the formation of the {Fe-(ferrozine)} complex, but not the{Fe-(bipyridine)} or the {Fe(II)-(phenanthroline)} complexes depend on the concentration of the iron chelator, requiring up to 20 min to complete when close to stoichiometric ratios are employed. The molar absorptivity values of these complexes under excess chelator concentrations were ~ 10% to 15% higher than reported literature values (i.e. 31,500 ± 1500 M cm for ferrozine at 562 nm, 9950 ± 100 M cm for 2,2'-bipyridine at 522 nm, and 12,450 ± 370 M cm for 1,10-phenanthroline at 510 nm). Our results have important implications when quantifying iron in biological systems and reveal optimal experimental conditions that must be employed for the accurate measurements of ferrous ions, whether free in solution, or after reduction of protein-bound ferric ions.
铁是几乎所有生命形式的必需营养素。由于其氧化还原性质以及在广泛的生物过程中的参与，已经开发出许多定性和定量的化学工具来检测生物分子中还原（Fe）和氧化（Fe）形式的铁。这些类型的测量不仅在检测溶液中的铁物种方面很重要，而且在理解铁在生理和病理过程中的分布、积累和作用方面也很重要。在这里，我们使用紫外可见分光光度法和三种常见的显色试剂，即铁嗪、2,2'-联吡啶和 1,10-菲啰啉，来检测和定量水溶液中亚铁离子的浓度，这是由于这些 Fe-螯合剂配合物的独特吸收光谱、特定摩尔吸光率和特征颜色。我们的结果表明，{Fe-（铁嗪）}配合物的形成动力学，但不是{Fe-（联吡啶）}或{Fe（II）-（菲啰啉）}配合物的形成动力学，取决于铁螯合剂的浓度，当接近化学计量比时，需要长达 20 分钟才能完成。在过量螯合剂浓度下，这些配合物的摩尔吸光率值比文献报道的值高约 10%至 15%（即在 562nm 处铁嗪为 31500±1500Mcm，在 522nm 处 2,2'-联吡啶为 9950±100Mcm，在 510nm 处 1,10-菲啰啉为 12450±370Mcm）。当在生物系统中定量铁时，我们的结果具有重要意义，并揭示了必须采用的最佳实验条件，以准确测量游离在溶液中的亚铁离子或还原蛋白结合的三价铁离子后的亚铁离子。
