Ferrous sulfate heptahydrate was purchased from J.T. Baker Chemical (Radnor, PA, USA), MOPS from Sigma-Aldrich (St. Louis, MO, USA) FMN and FAD from Cayman Chemical Company (Ann Arbor, MI, USA), NADPH from EMD Millipore Corporation (Burlington, MA, USA), riboflavin from TCI Chemicals (Tokyo, Japan), and ferrozine from Alfa Aesar (Haverhill, MA, USA).
Ferrozine
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ferrozine is a water-soluble iron chelating agent used for the colorimetric determination of iron in various sample types. It forms a stable, purple-colored complex with ferrous iron that can be quantified spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Folin ciocalteu s phenol reagent, by Merck Group (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Dpph 2 2 diphenyl 1 picrylhydrazyl, by Thermo Fisher Scientific (2 mentions)
Gallic acid, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Bromocresol purple, by Carl Roth (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Nadph, by Merck Group (1 mentions)
Ninhydrin, by Merck Group (1 mentions)
Zinc nitrate hexahydrate, by Merck Group (1 mentions)
Agar, by BD (1 mentions)
Nutrient broth, by BD (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
Ferrous chloride tetrahydrate, by Avantor (1 mentions)
Ferric chloride hexahydrate, by Merck Group (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
Potassium dihydrogen orthophosphate, by Thermo Fisher Scientific (1 mentions)
Ferric chloride hexahydrate, by Thermo Fisher Scientific (1 mentions)
Nutrient broth, by Thermo Fisher Scientific (1 mentions)
13 protocols using ferrozine
1
Ferrous Sulfate Heptahydrate Characterization
2
Porcine Skin Gelatin Extraction and Antioxidant Analysis
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GT from porcine skin (type A, bloom# 300), OA, GA, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu’s phenol reagent, zinc nitrate hexahydrate, and ninhydrin were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Ferrozine, EDC, and NHS were purchased from Alfa Aesar Co. (Ward Hill, MA, USA). Ferric chloride hexahydrate was acquired from Merck (Darmstadt, Germany). Potassium ferricyanide and trichloroacetic acid were purchased from Ferak Berlin GmbH (Berlin, Germany). Ferrous chloride tetrahydrate was obtained from J. T. Baker (Center Valley, PA, USA). Tryptic soy broth, nutrient broth, and agar were acquired from Difco Laboratories (Detroit, FL, USA).
3
Phytochemical and Antioxidant Analysis
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The various reagents used throughout this project were purchased from suppliers as follows. TPC analysis: Folin-Ciocalteu’s phenol reagent (2N, R and M Chemicals, Essex, UK), gallic acid (98%, Fluka, Steinheim, France), anhydrous sodium carbonate (99%, J. Kollin, UK), diphenyl-2-picrylhydrazyl (DPPH) assay: 2,2-diphenyl-1-picrylhydrazyl (90%, Sigma, St. Louis, MO, USA), ferric reducing power (FRP) assay: ferric chloride hexa-hydrate (100%, Fisher Scientific, Loughborough, UK), potassium ferricyanide (99%, Unilab, Auburn, Australia), trichloroacetic acid (99.8%, HmbG Chemicals, Barcelona, Spain), potassium dihydrogen orthophosphate (99.5%, Fisher Scientific), dipotassium hydrogen phosphate (99%, Merck, Darmstadt, Germany), iron chloride (99%, RandM Chemicals), ferrous ion chelating (FIC) assay: ferrozine (98%, Acros Organics, Morris Plains, NJ, USA), ferrous sulphate hepta-hydrate (HmbG Chemicals), ethylenediaminetetraacetic (99.5%, Bendosen Laboratory Chemicals, Bendosen, Norway), potassium acetate (99%, R and M chemicals), rutin (98%, Sigma), phytochemical screening: sulfuric acid (95%–97%, HmBG Chemicals), hydrochloric acid (37%, Merck, Darmstadt, Germany), Dragendorff reagent (Fluka), α-naphthol (99%, Sigma), antimicrobial activity: nutrient broth (Oxoid, Hampshire, England), nutrient agar (Oxoid, Hampshire, England), and vancomycin (Sigma).
4
Polished Rice and Herbal Compound Characterization
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Polished rice (11.9% moisture, 82.4% carbohydrate, 5.7% protein, and 0% lipid) from the 2017 crop was purchased from the Hua-Tung Rice Co., Ltd. (Hualien, Taiwan). Dried P. cuspidatum roots were purchased from local Chinese herbal medicine stores and ground into powder with ~0.62 mm particles. Qu was purchased from Yong Xin Jiuqu Co., Ltd. (Changhua, Taiwan). Piceid and p-nitrophenyl-β-D-glucopyranoside (PNG) was purchased from Sigma-Aldrich (MO, USA). Resveratrol was purchased from Changsha Nutramax Biotechnology (Changsha, China). DPPH (2,2-Diphenyl-1-picrylhydrazyl) and o-phthaldialdehyde were purchased from Alfa Aesar (Tewksbury, MA, USA). Finally, 3,5-dinitrosalicylic acid and ferrozine were purchased from Acros (Morris Plains, NJ, USA). Unless otherwise noted, all reagents and chemicals were of analytical grade.
5
Spectrophotometric Iron(II) Assay Protocols
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Esculetin
(6,7-dihydroxycoumarin, C9H6O4, >98%,
Alfa Aesar), scopoletin (6-methylesculetin,
C10H8O4, >99%, Sigma-Aldrich),
fraxetin
(7,8-dihydroxy-6-methoxycoumarin, C10H8O5, >98%, Sigma-Aldrich), iron(II) chloride tetrahydrate
(FeCl2·4H2O, >99%, Sigma-Aldrich), iron(III)
chloride
hexahydrate (FeCl3·6H2O, >99%, Merck),
Ferrozine (C20H13N4NaO6S2·xH2O, >98%, ACROS
organics), ammonium acetate (CH3COONH4, >98%,
Merck), sodium hydroxide (NaOH, >99%, Merck), hydrochloric acid
(HCl,
30% supra pure grade, Merck), and sodium chloride (NaCl, >99.5%,
Merck)
were used as received without further purification. Piperazine-1,4-bis(propane-sulfonic
acid) (PIPPS, C10H22N2O6S2, pKa1 = 3.73, pKa2 = 7.96, >97% pure, Merck), 2-morpholinoethanesulfonic
acid monohydrate (MES, C6H14N2·H2O, pKa = 6.06, >99%, Merck),
3-(N-morpholino)propanesulfonic acid (MOPS, C7H15NO4S, pKa = 7.20,
>99%, Carl Roth GmbH + Co), 1,4-dimethylpiperazine (DEPP, C6H14N2, pKa1 = 4.48,
pKa2 = 8.58, >98% Sigma-Aldrich), and N,N,N′,N′-tetramethylethylenediamine (TEEN, (CH3)2NCH2CH2N(CH3)2, pKa1 = 6.58, pKa2 = 9.88, >99.5%, Sigma-Aldrich) were used as pH buffers.
All experimental and analytical solutions were prepared with ultrapure
water (resistivity > 18.2 MΩ·cm, TOC < 2 ppb, Milli-Q,
Millipore).
(6,7-dihydroxycoumarin, C9H6O4, >98%,
Alfa Aesar), scopoletin (6-methylesculetin,
C10H8O4, >99%, Sigma-Aldrich),
fraxetin
(7,8-dihydroxy-6-methoxycoumarin, C10H8O5, >98%, Sigma-Aldrich), iron(II) chloride tetrahydrate
(FeCl2·4H2O, >99%, Sigma-Aldrich), iron(III)
chloride
hexahydrate (FeCl3·6H2O, >99%, Merck),
Ferrozine (C20H13N4NaO6S2·xH2O, >98%, ACROS
organics), ammonium acetate (CH3COONH4, >98%,
Merck), sodium hydroxide (NaOH, >99%, Merck), hydrochloric acid
(HCl,
30% supra pure grade, Merck), and sodium chloride (NaCl, >99.5%,
Merck)
were used as received without further purification. Piperazine-1,4-bis(propane-sulfonic
acid) (PIPPS, C10H22N2O6S2, pKa1 = 3.73, pKa2 = 7.96, >97% pure, Merck), 2-morpholinoethanesulfonic
acid monohydrate (MES, C6H14N2·H2O, pKa = 6.06, >99%, Merck),
3-(N-morpholino)propanesulfonic acid (MOPS, C7H15NO4S, pKa = 7.20,
>99%, Carl Roth GmbH + Co), 1,4-dimethylpiperazine (DEPP, C6H14N2, pKa1 = 4.48,
pKa2 = 8.58, >98% Sigma-Aldrich), and N,N,N′,N′-tetramethylethylenediamine (TEEN, (CH3)2NCH2CH2N(CH3)2, pKa1 = 6.58, pKa2 = 9.88, >99.5%, Sigma-Aldrich) were used as pH buffers.
All experimental and analytical solutions were prepared with ultrapure
water (resistivity > 18.2 MΩ·cm, TOC < 2 ppb, Milli-Q,
Millipore).
6
Detailed Reagents for Antioxidant Assays
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Acetic acid (>99%), methanol (>99%), ethanol (99.8%), sodium carbonate (Na2CO3) (99.5%) and Folin-Ciocalteu’s reagent (2.0 N) were purchased from Fisher Chemical (Fair Lawn, NJ, USA). Acetic acid potassium (99%), aluminum chloride hexahydrate (AlCl3·6H2O) (99%), EDTA (99%), ferrozine, gallic acid (98%), iron (II) chloride tetrahydrate (FeCl2·4H2O) and iron (III) chloride hexahydrate (FeCl3·6H2O) were supplied by Acros (Geel, Belgium). DPPH (2,2-diphenyl-1-picrylhydrazyl) (95%) and p-anisidine (99%) were purchased from Alfa Aesar (Haverhill, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.
7
Fe(III) Chelate Reductase and Acidification Assays
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The FeIII chelate reductase activity and acidification assays were conducted as described by Yi and Guerinot (1996) . For the acidification assay, seedlings were transferred after 3 days of treatment on Bromocresol media [Bromocresol Purple (0.006% w/v; Carl Roth, https://www.carlroth.com); CaSO 4 (0.2 mM); agar (Agar Select 0.8% w/v; Sigma-Aldrich); pH 6.5] for 18 h. For the FeIII chelate reductase activity assays, seedlings were pooled (5 by 5) and roots were immersed in the reductase solution Carl Roth) ; FerroZine (0.3 mM; Acros Organics, https:// www.acros.com)] for 20 min in the dark. The absorbance of the FeII-FerroZine complex formed in the solution was measured at 562 nm. The root weight of the sample and the molar extinction coefficient of the complex (28.6 mM -1 cm -1 ) were used for the final calculation.
8
Comprehensive Analytical Reagent Protocol
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Chemicals, including 2,2-diphenyl-1-picrylhydrazyl radical (DPPH; S7670), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ; T1253), ferric chloride (236489), sodium acetate trihydrate (S7670), L-ascorbic acid (A7506), salicylic acid (247588), sodium hydroxide (221465), potassium nitrate (P6083), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 113784), 2,2’,2’’,2’’’-(Ethane-1,2-diyldinitrilo)tetra acetic acid (EDTA) disodium salt dihydrate (ED2SS), L-cysteine (168149), sulphanilamide (S9251), and N-1-Naphhthylethylenediamine dihydrochloride (N9125), were obtained from Sigma-Aldrich (St Louis, Mo). Cupric sulphate pentahydrate (CuSO4.5H2O; BP346-500), methanol, hydrochloric acid, sulfuric acid, and glacial acetic acid were obtained from Fisher Scientific (USA). Trolox ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; 218940050), ferrozine (3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5’,5’’-disulfonic acid disodium salt; 410570010), and pyrocatechol violet (3,3’,4-trihydroxyfuchsone-2’’-sulfonic acid; 146540050) were obtained from Acros Organics (Morris, NJ, USA). Water was purified with a MilliQ filtration system. All reagents were of analytical grade purity. Handling of chemicals and discarding of waste were done in accordance with safe lab procedures using a chemical hood and appropriate protective gear.
9
Measuring Root Acidification and Ferric Chelate Reductase
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For the measure of acidification capacity, roots from a pool of 5 seedlings were incubated in 0.005% bromocresol purple (Roth) during 24 hours in the dark. A 433 of the protonated form of was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted April 5, 2021. ; https://doi.org/10.1101/2021.04.02.438214 doi: bioRxiv preprint the dye was then measured and expressed relative to the root weight of sample (Santi and Schmidt, 2009; El-Ashgar et al., 2012) .
The ferric chelate reductase (FCR) activity was measured on roots from a pool of 5 seedlings. Samples were immerged in a reductase solution containing FeIII-EDTA (0.1 mM, Roth) and FerroZine (0.3 mM, Acros Organics) for 30 minutes in the dark. A 562 of the FeII-FerroZine complex was then determined. The final calculation included the root weight of sample and the molar extinction coefficient of the complex (28.6 mM -1 .cm -1 ) (Yi and Guerinot, 1996) .
The copyright holder for this preprint (which this version posted April 5, 2021. ; https://doi.org/10.1101/2021.04.02.438214 doi: bioRxiv preprint the dye was then measured and expressed relative to the root weight of sample (Santi and Schmidt, 2009; El-Ashgar et al., 2012) .
The ferric chelate reductase (FCR) activity was measured on roots from a pool of 5 seedlings. Samples were immerged in a reductase solution containing FeIII-EDTA (0.1 mM, Roth) and FerroZine (0.3 mM, Acros Organics) for 30 minutes in the dark. A 562 of the FeII-FerroZine complex was then determined. The final calculation included the root weight of sample and the molar extinction coefficient of the complex (28.6 mM -1 .cm -1 ) (Yi and Guerinot, 1996) .
10
Antioxidant and Metal Chelation Assays
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2,2-diphenyl-1-picrylhydrazyl (DPPH), 2-deoxy-D-ribose 97% (Sigma-Aldrich, USA); acetic acid (R&M, UK); bovine serum albumin (BSA) (Sigma-Aldrich, USA); butylated hydroxytoulene (BHT), chloroform, ethylenediaminetetraacetic acid (EDTA) 99% (Sigma-Aldrich, USA); ferrozine 98% (Acros Organic, Belgium); Folin-Ciocalteu's phenol reagent (Merck, USA); gallic acid 98% (Acros Organic, Belgium); glacial acid (Riendemann Chmidt Chemical, Malaysia);hydrogen peroxide (Systerm, Malaysia); iron (II), iron (III) chloride (Riendemann Chmidt Chemical, Malaysia); N-1-naphthyl ethylene diamine dihydrochloride (R&D, UK); potassium ferricyanide (Labjax Chemical Ltd, India); quercitin hydrate (Aldrich Chemistry, USA); Sodium Dodecyl Sulfate (SDS) (Bio-Rad, USA); sodium hydroxide (NaOH), sodium nitroprusside, sulfanilic acid (Merk, USA); Thiobarbituric Acid (TBA), Trichloroacetic Acid (TCA) (Fisher Scientific, USA)
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