Ferrozine reacts with divalent iron to form a stable magenta complex species and used for the direct determination of iron in water with maximum absorbance at 562 nm [1,2]. The visible absorption spectrum of the ferrous complex of ferrozine exhibits a single sharp peak at 562 nm. At this wavelength, the molar absorptivity is 27,900 and the Beer-ambert law is obeyed to approximately 4 mg/L of Fe [1].
Ferrozine assay of ISE6 cells[3]
The ferrozine assay for measuring non-haem iron was adapted to determine the concentration of iron in ISE6 cells. After knockdown and/or iron exposure, cells were collected, and cell lysates were collected using the method described above. Concentrated HCl (99.5%) was added and then heated to 95 °C. After cooling to room temperature, the mixture was centrifuged, and the supernatant was obtained, to which was added 75 mM ascorbate or water. Afterward, 10 mM ferrozine was added. Saturated ammonium acetate was added to facilitate colour development. Absorbance was measured at 550 nm, and the iron concentration was calculated based on a molar extinction coefficient of the iron-ferrozine complex of 27,900 M-1cm-1 and based on the protein concentration. The protein concentration was measured using a Micro BCA Protein Assay Kit. The total iron concentration is computed from samples with ascorbate. The ferrous iron concentration was computed from samples without ascorbate (reducing agent), while the ferric iron concentration is computed from the difference between the total iron and ferrous iron concentration.
References:
[1]. Stookey L L. Ferrozine---a new spectrophotometric reagent for iron[J]. Analytical chemistry, 1970, 42(7): 779-781.
[2]. Jeitner T M. Optimized ferrozine-based assay for dissolved iron[J]. Analytical biochemistry, 2014, 454: 36-37.
[3]. Hernandez E P, Kusakisako K, Talactac M R, et al. Induction of intracellular ferritin expression in embryo-derived Ixodes scapularis cell line (ISE6)[J]. Scientific reports, 2018, 8(1): 1-12.
Ferrozine 与二价铁反应形成稳定的品红色络合物，用于直接测定水中的铁，最大吸光度在 562 nm [1,2]。亚铁锌络合物的可见吸收光谱在 562 nm 处显示出单个尖峰。在此波长下，摩尔吸光率为27,900，且Fe[1]约为4 mg/L时遵守比尔-安伯特定律。
ISE6 细胞的 Ferrozine 测定[3]
用于测量非血红素铁的亚铁嗪测定法适用于测定 ISE6 细胞中的铁浓度。在击倒和/或铁暴露后，收集细胞，并使用上述方法收集细胞裂解物。添加浓 HCl (99.5%)，然后加热至 95 °C。冷却至室温后，将混合物离心，得到上清液，向其中加入75mM抗坏血酸盐或水。之后，加入 10 mM 亚铁嗪。添加饱和乙酸铵以促进显色。在 550 nm 处测量吸光度，铁浓度基于 27,900 M-1cm-1 的铁-亚铁肼络合物的摩尔消光系数和基于蛋白质浓度计算。使用 Micro BCA 蛋白质测定试剂盒测量蛋白质浓度。总铁浓度是从含有抗坏血酸的样品中计算出来的。亚铁浓度由不含抗坏血酸（还原剂）的样品计算得出，而三价铁浓度由总铁浓度与亚铁浓度之差计算得出。