Ferrozine---a new spectrophotometric reagent for iron

Stookey, Lawrence

doi:10.1021/ac60289a016

Cited by 4,203 publications

(2,679 citation statements)

References 12 publications

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“…The concentrations of His ββ′ and FLAG ββ′ were determined using an ∊ 280-310 of 99 800 M −1 cm −1 and is reported as a concentration of monomer β or dimer ββ′ (28). Iron concentrations were determined by the ferrozine assay (34).…”

Section: Methodsmentioning

confidence: 99%

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

et al. 2006

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The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y • ) cofactor. In Saccharomyces cerevisiae, two R1s, α n and , have been identified, while R2 is a heterodimer (ββ′). β′ cannot bind iron and generate the Y • ; consequently, the maximum amount of Y • per ββ′ is 1. To determine the cofactor stoichiometry in vivo, a FLAGtagged β ( FLAG β) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of FLAG ββ′ by immunoaffinity chromatography, which was found to have 0.45 ± 0.08 Y • / FLAG ββ′ and a specific activity of 2.3 ± 0.5 μmol min −1 mg −1 . FLAG ββ′ isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y • / FLAG ββ′ ratio of 0.5. To determine the Y • /ββ′ ratio without R2 isolation, whole cell EPR and quantitative Western blots of β were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y • /ββ′ ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Δ gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y • /ββ′ ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y • and modulation of Y • concentration is not involved in the regulation of RNR activity.

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Section: Methodsmentioning

confidence: 99%

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

et al. 2006

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“…Soluble Fe was measured before and after sub-culturing by taking liquid samples anoxically using an N 2 flushed syringe, filtered through a 0.2 mm regenerated cellulose filter (Sartorius, G€ ottingen Germany) and transferred into 0.5 M HCl (trace metal grade, HN53.1, Carl Roth, Karlsruhe, Germany) (Sztejrenszus et al 2016). Fe 2C was measured following the ferrozine assay (Stookey 1970;Hegler et al 2008) with modifications (50% w:v ammonium acetate, 0.1% w:v ferrozine).…”

Section: Incubation Experimentsmentioning

confidence: 99%

Potentially Active Iron, Sulfur, and Sulfate Reducing Bacteria in Skagerrak and Bothnian Bay Sediments

Reyes

Schneider

Thürmer

et al. 2017

Geomicrobiology Journal

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In many marine surface sediments, the reduction of manganese (Mn) and iron (Fe) oxides is obscured by sulfate reduction, which is regarded as the predominant anaerobic microbial respiration process. However, many dissimilatory sulfate and sulfur reducing microorganisms are known to utilize alternative electron acceptors such as metal oxides. In this study, we tested whether sulfate and sulfur reducing bacteria are linked to metal oxide reduction based on biogeochemical modeling of porewater concentration profiles of Mn 2C and Fe 2C in Bothnian Bay (BB) and Skagerrak (SK) sediments. Steady-state modeling of Fe 2C and Mn 2C porewater profiles revealed zones of net Fe (0-9 cm BB; »10 and 20 cm SK) and Mn (0-5 cm BB; 2-8 cm SK) species transformations. 16S rRNA pyrosequencing analysis of the in-situ community showed that Desulfobacteraceae, Desulfuromonadaceae and Desulfobulbaceae were present in the zone of Fereduction of both sediments. Rhodobacteraceae were also detected at high relative abundance in both sediments. BB sediments appeared to harbor a greater diversity of potential Fe-reducers compared to SK. Additionally, when the upper 10 cm of sediment from the SK was incubated with lepidocrocite and acetate, Desulfuromonas was the dominant bacteria. Real-time quantitative polymerase chain reaction (qPCR) results showed decreasing dsrA gene copy numbers with depth coincided with decreased Fe-reduction activity. Our results support the idea that sulfur and sulfate reducing bacteria contribute to Fe-reduction in the upper centimeters of both sediments.

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“…Ferrozine is water soluble and does not react with Fe3+. The ferrous complex of ferrozine has an absorbance maximum at 562 nm and a molar extinction coefficient of 27 900 (Stookey, 1970). The reaction mixture contained (in a final volume of 2 ml) : 1 ml cell extract, 12.5 pM iron as ferric dcitrate, ferric-nitrilotriacetic acid (ferric-NTA), ferric-EDTA or ferric chloride ; 250 pM ferrozine; 250 pM NAD, reduced form (NADH; Sigma).…”

Section: Introductionmentioning

confidence: 99%

Ferric-reductase activities in whole cells and cell fractions of Vibrio (Listonella) anguillarum

Mazoy

Lemos

1996

Microbiology

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The ability of Wibrio (Listonella) anguillamm strains from serotype groups 01 and 02 to reduce Fe3+ in the form of different chelates was investigated. All strains, grown in M9 minimal medium supplemented with 0.2?40 Casamino acids, reduced Fe3+ complexed by citrate, nitrilotriacetic acid and EDTA. In whole cells, the degree of reduction was dependent on the Fe3+ ligand and on the strain, with the greatest values corresponding to ferric dicitrate and serotype group 01 strains, respectively. The ferric-reductase activity increased, over the basal levels, when the cells were grown with iron added as ferric dicitrate, haemin or haemoglobin. All strains also reduced ferricyanide, a compound that is not transported into the bacterial cells. Ferricyanide reduction was also increased when the cells were grown in the presence of an iron source. All of the cell fractions (periplasm, membranes and cytoplasm) showed Fe3+-reducing activity, with the highest values observed in the presence of Mgz+, NADH and FAD in the assay buffer. Cytoplasmic ferricreductase could be visualized using native polyacrylamide or starch gel electrophoresis, whereas the periplasmic and membrane reductase(s) could only be detected on starch gels. The results indicate the presence of different ferric-reductase activities in V. anguillarum, which could be involved in the different iron-acquisition systems present in this micro-organism, i.e. siderophore-mediated systems and siderophore-independent mechanisms.

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Ferrozine---a new spectrophotometric reagent for iron

Cited by 4,203 publications

References 12 publications

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

Potentially Active Iron, Sulfur, and Sulfate Reducing Bacteria in Skagerrak and Bothnian Bay Sediments

Ferric-reductase activities in whole cells and cell fractions of Vibrio (Listonella) anguillarum

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