Kinetics of the complexation of iron(II) with ferrozine

Thompsen, James C.; Mottola, Horacio A.

doi:10.1021/ac00268a037

Cited by 72 publications

(55 citation statements)

References 15 publications

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“…Iron Dissociation Rates-The dissociation rate of the iron complex in NorR was estimated using competition between NorR-Fe(II) and ferrozine (34,35). An excess of ferrozine (final concentration, 4 mM) was added in a gas-tight Hamilton syringe to NorR and mutant preparations (8 -16 M in 100 mM TrisHCl, pH 8.5, 100 mM NaCl, 40% glycerol), and the rate of Fe(II)-ferrozine formation was monitored under anaerobic conditions at 565 nm.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the Nitric Oxide-sensing Non-heme Iron Center in the NorR Regulatory Protein

Tucker

D’Autréaux

Yousafzai

et al. 2008

Journal of Biological Chemistry

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The NorR regulatory protein senses nitric oxide (NO) to activate genes required for NO detoxification under anaerobic and microaerobic conditions in Escherichia coli. NorR belongs to the 54 -dependent family of transcriptional activators and contains an N-terminal regulatory GAF (cGMP phosphodiesterase, adenylate cyclase, FhlA) domain that controls the ATPase activity of the central AAA؉ domain to regulate productive interactions with 54 . Binding of NO to a non-heme iron center in the GAF domain results in the formation of a mononitrosyl-iron complex and releases intramolecular repression of the AAA؉ domain to enable activation of transcription. In this study, we have further characterized NorR spectroscopically and substituted conserved residues in the GAF domain. This analysis, in combination with structural modeling of the GAF domain, has identified five candidate ligands to the non-heme iron and suggests a model in which the metal ion is coordinated in a pseudo-octahedral environment by three aspartate residues, an arginine, and a cysteine.

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Section: Methodsmentioning

confidence: 99%

Analysis of the Nitric Oxide-sensing Non-heme Iron Center in the NorR Regulatory Protein

Tucker

D’Autréaux

Yousafzai

et al. 2008

Journal of Biological Chemistry

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“…The upper pH limit for the ferrozine-Fe(II) reaction was not fixed but reliant on the specific buffer choice; pH 6.5 (acetate buffer), pH 7 (phosphate), pH 7.5 (bicarbonate buffer), pH 8.0 (triethanolamine), pH 8.5 (tris-HCl) or pH 9,0 for borate buffer [44]. Past research demonstrated that ferrozine-Fe (II) complex was stable at pH 4-pH11 [27,44,54]. Finally, the ferrozine reagent used for this study was identical to (1.85mM Ferrozine plus 0.93mM iron (III)) chloride) that was adopted for autoanalyzer determination of plasma ascorbic acid in acetate buffer at pH 4.0 [21,38].…”

Section: General Issues For Frap Assay Formulations At Ph 70mentioning

confidence: 99%

A FRAP Assay at pH 7 unveils Extra Antioxidant Activity from Green, Black, White and Rooibos Tea but not Apple Tea

Apenten¹

2015

F Nutr Reprt

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Aim: Realization of a ferric reducing antioxidant power (FRAP) assay at neutral pH and re-evaluation of tea antioxidant activity for comparisons with the standard FRAP assay. Method:A FRAP assay at neutral pH utilized ferrozine (7.3-(2-Pyridyl)-5, 6-diphenyl-1, 2, 4-triazine-4' , 4''-disulfonic acid; ferrozine) dye in conjunction with Tris-HCl buffer (0.1M. pH 7.0) with 280 µl of regent addition to 20 µl of tea infusions and absorbance measurements at 562 nm with a microplate reader. Results:The microplate ferrozine FRAP assay (mFzFRAP) gave linear calibrations for ascorbic acid, gallic acid, ammonium ferrous sulphate, (AFS), trolox, cysteine and glutathione (R 2 = 0.998 -1.000) with molar absorptivity (measures of sensitivity) similar to literature values. The analytical precision was 5-7% and the minimum detectable concentrations (MDC) were 1.4-2.8 µM (0.4-0.8 nanomoles).Discussion: Values for FRAP were higher at pH 7.0 compared to pH 4.0 for gallic acid, ascorbic acid, glutathione, and cysteine possibly due to their ionization at high pH. The assay sensitivity for AFS and trolox were unchanged at pH 4.0 and pH 7.0. When assayed at pH 7 the water infusions from green tea, black tea, white tea, and rooibos tea had 200-360% antioxidant activity normally observable at low pH. Conclusion:A FRAP assay at pH 7 unveils extra antioxidant activity for green, black, white and Rooibos teas compared to values from the standard TptzFRAP (pH 3.6) method. As a recommendation, the antioxidant activity of teas and other herbal preparations should be re-evaluated over a wide pH range.

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“…As this topic alone is immense in scope, and most readers are perhaps already familiar with spectrophotometry, the reader is referred to any number of references on this subject (Thomas, 1996;Skoog et al, 2007;Kellner, 1998). Most metal ions such as iron are weakly absorbing species that can be made very strongly absorbing via complexation of iron ions with an appropriate complexing agent, which is usually selective for a particular valence of iron (i.e., Fe 2+ or Fe 3+ ) (McBryde, 1964;Stookey, 1970).…”

Section: Spectrophotometric Determinations Of Metal Ionsmentioning

confidence: 99%

“…As this chapter will focus on the stoichiometric aspects of the spectrophotometric determination of iron (as Fe 2+ ) using the clinical iron-marking chelators Ferene S and Ferrozine, one needs to know about Beer's Law (Thomas, 1996;Skoog et al, 2007;Kellner, 1998;Harris, 2010), which is perhaps most familiar as…”

Section: Spectrophotometric Determinations Of Metal Ionsmentioning

confidence: 99%

Limiting Reactants in Chemical Analysis: Influences of Metals and Ligands on Calibration Curves and Formation Constants for Selected Iron-Ligand Chelates

Stauffer¹,

Weller²,

Kubas³

et al. 2012

Stoichiometry and Research - The Importance of Quantity in Biomedicine

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Kinetics of the complexation of iron(II) with ferrozine

Cited by 72 publications

References 15 publications

Analysis of the Nitric Oxide-sensing Non-heme Iron Center in the NorR Regulatory Protein

Analysis of the Nitric Oxide-sensing Non-heme Iron Center in the NorR Regulatory Protein

A FRAP Assay at pH 7 unveils Extra Antioxidant Activity from Green, Black, White and Rooibos Tea but not Apple Tea

Limiting Reactants in Chemical Analysis: Influences of Metals and Ligands on Calibration Curves and Formation Constants for Selected Iron-Ligand Chelates

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