Hydrogen peroxide concentration was determined according to Batdorj et al. protocol [70 (link)] with slight modifications. Cells were harvested by centrifugation at 11500 g, 15 °C for 10 min. One hundred microliter of the sample supernatant were mixed with 100 μl of 4-aminoantipyrine 4 mg.ml− 1 (Sigma-Aldrich, St. Louis, Missouri, USA), 20 μl of water-saturated phenol (Sigma-Aldrich, St. Louis, Missouri, USA), 750 μl of phosphate buffer Na2HPO4/NaH2PO4 0.1 M (pH 7) and 30 μl of horseradish peroxidase type VI-A (500 U.ml− 1 in sodium phosphate buffer pH 6 (Sigma-Aldrich, St. Louis, Missouri, USA). Sample was mixed by inverting the tube and OD was measured at 505 nm. Blank was done by replacing sample by sterile medium. H2O2 concentration was determined using a standard curve performed with concentrations ranging from 0 to 3 mM. The minimal concentration that could be detected was 0.5 mM.
Horseradish peroxidase type 6 a
Manufactured by Merck Group
Sourced in United States
Horseradish peroxidase type VI-A is an enzyme isolated from the roots of the horseradish plant. It is a commonly used reagent in various biochemical and analytical applications due to its ability to catalyze the oxidation of substrates in the presence of hydrogen peroxide.
Automatically generated - may contain errors
Lab products found in correlation
Water saturated phenol, by Merck Group (1 mentions)
L amino acid, by Merck Group (1 mentions)
Phosphate citrate buffer, by Merck Group (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Spectramax l microplate reader, by Molecular Devices (1 mentions)
Luminol, by Merck Group (1 mentions)
White 96 well plate, by Greiner (1 mentions)
Horse heart cytochrome c, by Merck Group (1 mentions)
Potassium cyanide, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Potassium ferrocyanide, by Merck Group (1 mentions)
Superoxide dismutase, by Merck Group (1 mentions)
Potassium phosphate monobasic and dibasic, by Merck Group (1 mentions)
Dodecyl maltoside, by Anatrace (1 mentions)
Ym 100, by Merck Group (1 mentions)
Potassium hydroxide, by Merck Group (1 mentions)
Potassium ferricyanide, by Merck Group (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
6 protocols using horseradish peroxidase type 6 a
1
Hydrogen Peroxide Quantification Assay
2
LAAO Activity Assay for H2O2 Release
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LAAO activity was determined using 100 μg total cellular protein by measuring H2O2 release according to the method of Mason et al.[14 ]: 100 μl L-amino-acid (10 mM; Sigma) or a dilution of a known concentrations of H2O2 (1.47–1470 μM; Sigma) was added to individual wells in a 96-well plate. A fresh premix of 10 μl 200 U/ml horseradish peroxidase type VI-A, 10 μl 10 mg/ml o-phenylenediamine, and 20 μl 500 mM phosphate citrate buffer (pH 5.0; Sigma) was prepared and 60 μl of cell extract or a H2O2 control was added. The complete 100 μl premix was added to wells, and the 200-μl reaction mixture was incubated for 2 h at 37°C with atmospheric O2 in a humidified 5% CO2 incubator. The reaction was stopped by adding 11 μl 36 N H2SO4. Samples were centrifuged at 13,000×g for 10 min to pellet any insoluble material and soluble material was transferred to a fresh 96-well plate; the A490 measured using a microplate reader (Bio-Rad). The OD490 background reading from empty-vector transfectants was subtracted from that of pcDNA4-IL4I1 transfectants. To determine the specific LAAO activity (U/mg total protein), total protein was measured by using the BCA protein assay.
3
Luminol-Based ROS Detection in Plants
4
Isolation and Characterization of Cytochrome c Oxidase
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Potassium phosphate monobasic and dibasic, potassium hydroxide, potassium ferricyanide, potassium ferrocyanide, potassium sulfate, potassium cyanide, horse heart cytochrome c, superoxide dismutase (SOD), horseradish peroxidase type VI A, and catalase from bovine liver were purchased from Sigma-Aldrich, Triton X-100 (TX) was from Roche Diagnostics, dodecyl maltoside (DM) from Anatrace, Sepharose Q fast flow from Pharmacia Uppsala and hydrogen peroxide solution (~30%) was from Fluka.
Bovine heart cytochrome c oxidase was isolated from mitochondria following the modified method [86 (link)] into 10 mM Tris, pH 7.6, 50 mM K2SO4, and 0.1% TX. To change TX for DM detergent, the purified enzyme was diluted and reconcentrated using microfilters (YM 100, Millipore, cut-off 100 kDa) with the buffer containing 0.1% DM.
Isolated CcO was frozen in liquid nitrogen and stored at −80 °C. The concentration of CcO was determined from the UV-Vis absorption spectrum of the oxidized enzyme using an extinction coefficient ε (424 nm) = 156 mM−1cm−1 and ε (428 nm) = 169 mM−1cm−1 for the cyanide-ligated CcO (CcO.CN) [87 (link)].
Bovine heart cytochrome c oxidase was isolated from mitochondria following the modified method [86 (link)] into 10 mM Tris, pH 7.6, 50 mM K2SO4, and 0.1% TX. To change TX for DM detergent, the purified enzyme was diluted and reconcentrated using microfilters (YM 100, Millipore, cut-off 100 kDa) with the buffer containing 0.1% DM.
Isolated CcO was frozen in liquid nitrogen and stored at −80 °C. The concentration of CcO was determined from the UV-Vis absorption spectrum of the oxidized enzyme using an extinction coefficient ε (424 nm) = 156 mM−1cm−1 and ε (428 nm) = 169 mM−1cm−1 for the cyanide-ligated CcO (CcO.CN) [87 (link)].
5
Kinetics of Peroxidase-Catalyzed ETE-S Oxidation
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A hydrogen peroxide (Z30%, Sigma Aldrich) stock solution of 10 mM was prepared freshly before mixing with an ETE-S stock solution of 1.8 mM. Horseradish Peroxidase type VI-A (Sigma Aldrich) aliquots of 1 mg ml À1 (Z250 U ml À1 ) were freshly prepared before use and added to the mixture of H 2 O 2 /ETE-S. The final concentrations of the reagents were: ETE-S 360 mM, H 2 O 2 180 mM and HRP 5 U ml À1 . The pH of the solutions prior and after the reaction were between pH 5-6. The absorption spectra were recorded from 300 to 900 nm (step 10 nm) using a microplate reader (BioTek, Synergy H1). For the H 2 O 2 dependence investigation, different amounts of H 2 O 2 from 0.1 to 2 equivalent vs. ETE-S concentration of 360 mM were added. The plate was mixed for 30 s and spectra were acquired 24 hours after the preparation of the samples. HRP activity was kept constant at 5 U ml À1 . For the HRP dependence investigation, the molar ratio ETE-S/H 2 O 2 was kept constant at 0.5 with a concentration of ETE-S at 360 mM. The HRP activity was varied from 0.1 U ml À1 to 100 U ml À1 . The plate was mixed for 30 s and spectra were acquired 1 hour after the preparation of the samples.
6
Aerobic H2O2 Accumulation in Anaerobic Cultures
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To measure H 2 O 2 accumulation upon aeration of cultures grown under anaerobic conditions, we cultivated 100-ml BHI medium cultures of E. italicus and L. garvieae strains in 500-ml flasks at 37 °C without agitation in jar containers. Cultures were removed from the anaerobic conditions and aerated on an orbital shaker. To measure bacterial growth and H 2 O 2 accumulation in aerobic cultures, overnight cultures were grown without shaking, diluted into 100 ml of prewarmed BHI medium aerated at 37 °C and centrifuged at 10,000 g. The H 2 O 2 levels of bacterium-free culture eluates were measured with horseradish peroxidase type VI-A (Sigma) at 505 nm. The H 2 O 2 was quantified using standard curve determined with H 2 O 2 (Sigma) solutions at concentrations ranging from 1.5 to 100 mmol l -1 .
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