A Comparison of Bathophenanthrolinedisulfonic Acid and Ferrozine as Chelators of Iron(II) in Reduction Reactions

Cowart, Richard E.; Singleton, F.L.; Hind, J.S.

doi:10.1006/abio.1993.1246

Cited by 92 publications

(65 citation statements)

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“…For the Fe-citrate uptake experiments, a stock solution of 100 µmol/L 55 FeCl 3 was prepared in the presence of a 100-fold molar excess of citrate as described by Richardson and Ponka. 38 Reduction from Fe 3ϩ to Fe 2ϩ in the presence of ascorbate (1-20 minutes of incubation) was measured as described by Cowart et al 39 Briefly, Fe-NTA (final concentration 1-40 µmol/L) was added to phosphate buffered saline or medium containing 20 mmol/L HEPES, 1 mmol/L ascorbate, and 1.5 mmol/L ferrozine, and the absorbance was measured at 562 nm. The concentration of Fe 2ϩ was calculated by comparing the sample absorbance with that obtained for standards containing 1-60 µmol/L ferrous sulphate.…”

Section: Methodsmentioning

confidence: 99%

Nitric oxide reduces nontransferrin-bound iron transport in HepG2 cells

et al. 1999

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Nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) modulate iron regulatory protein (IRP) activity and may, therefore, affect iron uptake through transferrin receptor expression. However, iron also enters the cell as nontransferrin-bound iron (NTBI), and the aim of this study was to evaluate the effects of NO donors on NTBI transport in HepG2 cells, a model of liver physiology. Incubation with SNP and SNAP led to a time-and concentration-dependent reduction in Fe 3؉ and Fe 2؉ uptake, thus indicating an effect on the transporter rather than on the reductase. In terms of Fe 2؉ uptake, no variations in the Michaelis-Menten constant (K m ) and a reduction in maximum uptake (V max ) (50, 33, and 16.6 fmol/g protein/min in control, SNP-, and SNAPtreated cells, respectively) were detected, which suggested a decrease in the number of putative NTBI transport protein(s). Gel shift assays showed that IRP activity was reduced by SNP and slightly increased by SNAP. Northern blot analysis of transferrin receptor messenger RNA (mRNA) levels showed variations similar to those observed for IRPs, but both NO donors increased L-ferritin mRNA levels and had no effect on the stimulator of Fe transport (SFT) mRNA. In conclusion, NO donors significantly reduce NTBI transport in HepG2 cells, an effect that seems to be IRP and SFT independent. Moreover, the reduction in NTBI uptake after NO treatment suggests that this form of iron may play a minor role in the increased hepatic iron stores observed in inflammation or that other liver cells are more involved in this pathological condition. (HEPATOLOGY 1999;29:464-470.)It has been reported that hepatocytes and Kupffer cells both produce nitric oxide (NO) in inflammatory states, mainly because of the upregulation of an inducible form of nitric oxide synthase by cytokines and lipopolysaccharides. 1,2 NO has been implicated in the regulation of a number of cell processes, 3 including the transport of Na ϩ across the cell membranes in proximal renal tubules, 4 and of HCO 3Ϫ and glutathione in the liver. 5 NO also plays an important role in iron homeostasis, mainly by modulating the activity of iron regulatory proteins (IRPs). 6-8 IRP1 and IRP2 regulate ferritin and transferrin receptor expression upon binding to the iron regulatory elements (IRE) respectively located in the 5Ј and 3Ј untranslated regions of their messenger RNAs (mRNAs). This binding stabilizes transferrin receptor mRNA and prevents the translation of ferritin mRNA. 7,9,10 The effects of NO on IRPs are different 11-15 depending on the analyzed cell type and the kind of NO donor-generated radicals, such as NO ϩ and NO · . NO · may coordinate directly with the 4Fe-4S cluster of IRPs, 16 whereas NO ϩ nitrosylation of the thiol groups of IRPs has been hypothesized on the basis of evidence obtained in other proteins. 17 However, NO can alter intracellular iron metabolism not only by modulating transferrin-bound iron transport, 11 but also through other mechanisms such as the release of ir...

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Section: Methodsmentioning

confidence: 99%

Nitric oxide reduces nontransferrin-bound iron transport in HepG2 cells

et al. 1999

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“…However, no pH dependence was observed when bipyridyl was used as chelator, indicating that the pH dependence was an artifact of the chelator assay. Chelator effects on measuring Fe 3ϩ reduction have been noted previously (44). Therefore, we redesigned the release assay so that chelator was not present until the point at which we wished to observe the concentration of immediately available Fe 2ϩ .…”

Section: Reductant and Chelator Dependence Of Bfr Iron Releasementioning

confidence: 98%

A New Role for Heme, Facilitating Release of Iron from the Bacterioferritin Iron Biomineral

Yasmin

Andrews

Moore

et al. 2011

Journal of Biological Chemistry

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Bacterioferritin (BFR) fromEscherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe 3؉ mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe 2؉ chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.

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“…Various drawbacks have been identified with all available Fe(II) methods. It is relatively straightforward, for example, to demonstrate that the unmodified ferrozine method appears to over-estimate nanomolar Fe(II) concentrations in some natural waters (Figure 3; Murray and Gill, 1978;Box, 1984;Cowart et al, 1993), which could be attributed to reduction of labile Fe(III) phases in the presence of NOM after the ferrozine ligand is added. Short mixing times between ferrozine addition and absorbance measurement, combined with handling samples in the dark and buffering to near-neutral pH, is reported to minimize, although not eliminate, the interference due to labile Fe(III) reduction (Box, 1984;Pullin and Cabaniss, 2001).…”

Section: Potential Explanations For Inter-methods Differences In Fe(iimentioning

confidence: 99%

A Comparison between Four Analytical Methods for the Measurement of Fe(II) at Nanomolar Concentrations in Coastal Seawater

et al. 2017

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A Comparison of Bathophenanthrolinedisulfonic Acid and Ferrozine as Chelators of Iron(II) in Reduction Reactions

Cited by 92 publications

References 0 publications

Nitric oxide reduces nontransferrin-bound iron transport in HepG2 cells

Nitric oxide reduces nontransferrin-bound iron transport in HepG2 cells

A New Role for Heme, Facilitating Release of Iron from the Bacterioferritin Iron Biomineral

A Comparison between Four Analytical Methods for the Measurement of Fe(II) at Nanomolar Concentrations in Coastal Seawater

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