Complexation of ferrous ions by ferrozine, 2,2′-bipyridine and 1,10-phenanthroline: Implication for the quantification of iron in biological systems

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 (Fe²⁺) and oxidized (Fe³⁺) 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 Fe²⁺-chelator complexes. Our results show that the kinetics of the formation of the {Fe²⁺-(ferrozine)₃} complex, but not the{Fe²⁺-(bipyridine)₃} or the {Fe(II)-(phenanthroline)₃} 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⁻¹ cm⁻¹ for ferrozine at 562 nm, 9950 ± 100 M⁻¹ cm⁻¹ for 2,2′-bipyridine at 522 nm, and 12,450 ± 370 M⁻¹ cm⁻¹ 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.