Ferrozine
(Synonyms: 菲啰嗪一钠盐) 目录号 : GC47346
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 。
Cas No.:69898-45-9
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >97.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1、将细胞裂解物的等分试样（100μL）置于Eppendorf管中，并与100μL 10mM HCl（Fe3+的溶剂）和100μL铁释放试剂（使用超纯水溶解等体积的1.4M HCl和4.5%（w/v）KMnO4，新鲜配制）混合；
注：HCl/KMnO4预处理能够从蛋白质中定量释放铁，包括铁储存蛋白铁蛋白和血红素蛋白如血红蛋白。HCl/KMnO4介导的含铁蛋白质的消化对于血红素蛋白质的铁定量是必不可少的。例如，如果蛋白质未经酸性高锰酸盐溶液预处理，则用铁锌处理血红蛋白不会导致任何Fe2+- Ferrozine复合物的回收。
2、将混合物在60℃的通风橱中孵育2小时，因为反应过程中会产生氯气；
3、在混合物冷却至室温后，将30μL的铁检测试剂（6.5mM Ferrozine、6.5mM neocuproine、2.5M乙酸铵和1M抗坏血酸溶于水中）加入到每个管中；
4、30分钟后，将每个管中的280μL溶液转移到96孔板的孔中，并在微孔板读取器上在562nm处测量吸光度。
5、100μL FeCl3标准品（0-300μM）在10 mM HCl、100μL 50 mM NaOH、100μL释放试剂和30μL检测试剂中的混合物。通过将样品的吸光度与以类似于样品的方式制备的等体积标准浓度范围的吸光度进行比较来计算样品的铁含量。
注意事项：
1）荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
2）为了您的安全和健康，请穿实验服并戴一次性手套操作。
References:
[1]. Jan Riemer,et,al. Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. 2004 Aug 15;331(2):370-5. doi: 10.1016/j.ab.2004.03.049.
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。
| Cas No. | 69898-45-9 | SDF | |
| 别名 | 菲啰嗪一钠盐 | ||
| Canonical SMILES | OS(C1=CC=C(C2=C(C3=CC=C(S([O-])(=O)=O)C=C3)N=C(C4=CC=CC=N4)N=N2)C=C1)(=O)=O.[Na+] | ||
| 分子式 | C20H13N4O6S2.Na | 分子量 | 492.5 |
| 溶解度 | PBS (pH 7.2): 1 mg/ml | 储存条件 | Store at 2-8°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.0305 mL | 10.1523 mL | 20.3046 mL |
| 5 mM | 0.4061 mL | 2.0305 mL | 4.0609 mL |
| 10 mM | 0.203 mL | 1.0152 mL | 2.0305 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells
Anal Biochem 2004 Aug 15;331(2):370-5.PMID:15265744DOI:10.1016/j.ab.2004.03.049.
The ferrozine-based colorimetric assay described here permits the quantitation of iron in cultured cells in amounts ranging between 0.2 and 30 nmol. Ferrous and ferric iron were detected equally well by the assay and the accuracy was unaffected by other divalent metal cations. This colorimetric assay was used to study iron accumulation in brain astrocytes that had been cultured in 24-well dishes. Iron complexed to cellular proteins was made accessible to Ferrozine by treatment of cell lysates with acidic KMnO(4) solution. The basal amounts of iron in untreated astrocyte cultures were approximately 10 nmol iron per mg protein. Incubation of the cells with ferric ammonium citrate caused the total cellular iron content to increase in a concentration-dependent manner. The estimates of cellular iron content that were obtained with the ferrozine-based assay did not differ from those determined by atomic absorption spectroscopy. The colorimetric assay described here provides a sensitive, cheap, and reliable method for the quantitation of intracellular iron and for the investigation of iron accumulation in cultured cells.
Optimized ferrozine-based assay for dissolved iron
Anal Biochem 2014 Jun 1;454:36-7.PMID:24632099DOI:10.1016/j.ab.2014.02.026.
The following report describes a simple and optimized assay for the detection of iron in solution based on the binding of this metal by Ferrozine. This assay accurately measures between 1 and 200 μM sample iron concentrations within 2½ hours.
Complexation of ferrous ions by Ferrozine, 2,2'-bipyridine and 1,10-phenanthroline: Implication for the quantification of iron in biological systems
J Inorg Biochem 2021 Jul;220:111460.PMID:33866045DOI:10.1016/j.jinorgbio.2021.111460.
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.
Antioxidant activity of Ferrozine-iron-amino acid complexes
Proc Natl Acad Sci U S A 2001 Jan 16;98(2):451-6.PMID:11149957DOI:10.1073/pnas.98.2.451.
Amino acid-Fe(II)-chelator complexes exhibit strong antioxidant activity. Taking advantage of the unique spectral characteristics of the complexes formed when Ferrozine (Fz) is used as the chelator, we now show that the primary blue complex (epsilon(max) at 632 nm) decomposes by two independent pathways: (i) a nonoxidative pathway involving dissociation of the amino acid component and formation of a purple complex (epsilon(max) at 562 nm) and (ii) an oxidative pathway leading to Fe(III) and colorless products. Quantitative conversion of the blue to purple complex yields an isosbestic point (i.p.) at 601 nm, whereas no i.p. is formed during quantitative oxidation of the blue complex. However, under some experimental conditions, decomposition of the blue product occurs by both pathways, leading to occurrence of a clean i.p. at wavelengths varying from 601 to 574 nm. Results of simulation experiments, confirmed by direct analysis, demonstrate that shifts in the i.p. reflect differences in the fractions of blue compound that decompose by the oxidative and nonoxidative pathways. Indeed, the fraction of blue that is converted to the purple complex is readily deduced from the wavelength of the i.p. These results suggest that identification of a physiological chelator that can replace Ferrozine in amino acid-iron complexes might have important physiological and pharmacological applications.
A Production-Accessible Method: Spectrophotometric Iron Speciation in Wine Using Ferrozine and Ethylenediaminetetraacetic Acid
J Agric Food Chem 2019 Jan 16;67(2):680-687.PMID:30561197DOI:10.1021/acs.jafc.8b04497.
Wine oxidation is reported to be linked to the iron species present in the wine, but spectrophotometric speciation is plagued by unstable measurements due to alterations to the reduction potential of iron by complexing agents. Ferrozine raises the reduction potential of iron by complexing preferentially to iron(II), inducing the reduction of iron(III) during analysis; here, EDTA is added to chelate iron(III) and to stabilize the forms of iron. Bisulfite addition allows the use of Ferrozine for red wine analysis by mitigating color interference. Measurements agree with values from a previous method for iron(II) and from FAAS for total iron. Spike recoveries were in the range of 103.5-110.1%. The method is linear for iron concentrations in the range of 0.10-6.00 mg L-1 and offers good precision (CV 0.4-10.1%) and low limits of detection (0.02 mg L-1) and quantification (0.06 mg L-1). The method demonstrated changes to iron speciation during the oxygenation of red wines.