The hydroxyethyldisulfide (HED, Sigma-Aldrich) reduction activity was measured at RT by following the NADPH oxidation in the presence of the GRX reducing system. The 100 μL reaction mixture contained 200 µM NADPH, 2 mM GSH, 0.7 mM HED, 5 mM EDTA and 1 µM GR in 100 mM Tris-HCl, pH 7.5. After 5 min of preincubation at 25 °C, a baseline was recorded before 0.1 µM GRX was added to start the reaction. The decrease in absorbance was measured spectrophotometrically at 340 nm (Cary 3500 UV-Vis, Agilent, Santa Clara, CA, USA). Enzyme activities were calculated using a molar ε-value for NADPH of 6220 M−1.cm−1.
Cary 3500 uv vis
Manufactured by Agilent Technologies
Sourced in United States
The Cary 3500 UV-Vis is a high-performance spectrophotometer designed for accurate and reliable absorbance measurements across a wide range of applications. It features a dual-beam optical system, a wavelength range of 185 to 900 nm, and advanced optics for precise data acquisition.
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Lab products found in correlation
Phenol red, by Carl Roth (2 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Cary 630 ftir spectrometer, by Agilent Technologies (1 mentions)
Malvern zetasizer nano z, by Malvern Panalytical (1 mentions)
Cary 3500 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
9 protocols using cary 3500 uv vis
1
Glutaredoxin-Mediated NADPH Oxidation Assay
2
Quantification of Thiol-Peroxidase Activity
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The rate of H2O2 reduction by thiol-peroxidases was quantified using the TRX system as reductant in the case of GPXL8 and the GRX system in the case of PRXIIB. The activity was measured by monitoring NADPH oxidation by spectrophotometry at 340 nm (Cary 3500 UV-Vis, Agilent, Santa Clara, CA, USA). The assay was performed in quartz cuvettes with 3 μM thiol peroxidase, 200 μM NADPH and 5 mM EDTA in 0.1 M Tris-HCl, pH 7.5, at RT. Varying amounts of TRX in combination with 1 µM NTRA were tested for optimizing the TRX system. For optimizing the GRX system, 2 mM GSH and 1 µM GR were supplemented with varying amounts of GRX. The reaction mixture (100 µL) was incubated at 25 °C for 5 min. OPDA was added and incubation time adjusted as indicated. The assay was equilibrated for 3 min, the baseline recorded, the reaction initiated by the addition of 300 µM H2O2 and the absorption change monitored at 340 nm. Enzyme activities were calculated using an ε-value for NADPH of 6220 M−1.cm−1.
3
Measurement of βCA1 Activity
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Activity of βCA1 was measured according to [22 (link),23 (link)]. Recombinant His6-tagged βCA1 was pretreated with indicated DTT concentrations for 30 min at room temperature. For the reoxidizing experiment, reduced βCA1 was desalted using Zeba Spin Desalting Columns (Thermo Fisher Scientific, Waltham, MA, USA) prior to oxidation with indicated substances. The βCA1 protein was added to 2 mL 20 mM Tris-HCl (pH 8.5 at 5 °C) with 15 µg/mL phenolred (T127.2, Carl Roth, Karlsruhe, Germany) at a final concentration of 50 nM βCA1. Then, 1.33 mL of ice-cold CO2-saturated water with 15 µg/mL phenolred was added to the cuvette to start the reaction at 2 °C. The reaction was monitored spectrophotometrically at 558 nm as decrease of absorbance (Cary 3500 UV-Vis, Agilent, Santa Clara, CA, USA). The Wilbur-Anderson Unit (WAU) [22 (link),24 ] was determined as the time span required for the pH value to drop from 8.3 to 6.3. To determine the enzyme activity, ex vivo proteins were extracted from leaves. Pulverized plant material was mixed with 20 mM Tris-HCl (pH 8.5 at 5 °C), centrifuged at 13,000× g at 4 °C for 5 min, and protein content of the supernatant was determined using the Bradford assay [25 (link)]. Defined amounts of protein were subjected to the βCA1 activity measurement without prior reduction with DTT.
4
NADPH Oxidation Kinetics in Reconstituted System
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NADPH oxidation was monitored in the reconstituted system after the addition of 100 μM H2O2 by spectrophotometry at 340 nm (Cary 3500 UV–Vis, Agilent, Santa Clara, CA, USA). The reconstitution mix contained 0.35 μM GPXL2, 0.15 μM GPXL8, 0.16 μM NTRA, 0.15 μM GR, 0.15 μM TRXh1, 0.15 μM TRXh2, 1.59 μM TRXh3, 0.71 μM TRXh5, 0.15 μM GRXC1, 1.02 μM GRXC2, 0.95 μM PRXIIB, 0.15 μM PRXIID, 0.15 μM TDX, 7.42 μM GAPC2 and 2.56 μM MDH1, supplemented with 500 μM GSH and 200 μM NADPH in 40 mM KPi, pH 7.2. Sample equilibration occurred at 21 °C and 500 rpm for 60 min and storage on ice. After recording the basal absorbance of 95 μl master mix at 340 nm for 4 min, addition of 5 μl 2 mM H2O2 initiated the reaction, i.e., 100 μM H2O2 final concentration or a pulse of 10 nmol H2O2. The recording proceeded for additional 25 min either in the complete set or in the absence of single components as indicated. NADPH oxidation was calculated based on ε = 6220 M−1cm−1.
5
GAPC2 Activity Assay with OPDA
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Recombinant His10-tagged GAPC2 (30 µM) was incubated with 140 µM NAD+ and 5 mM DTT at RT for 30 min. In total, 1 µM reduced and desalted (Zeba Spin Desalting Columns, Thermo Fisher Scientific, Waltham, MA, USA) GAPC2 and 140 µM NAD+ were mixed with 25, 50, 100 or 500 µM OPDA or the respective concentrations of solvent (EtOH), 50 µM H2O2 or 10 mM DTT, followed by a 1 h incubation at RT.
The reaction mixture containing 4 mM ATP, 8 mM 3-phosphoglyceric acid disodium salt (3-PGA), 0.294 mM NADH, 90 nM phosphoglycerate kinase (PGK), 8 mM MgSO4 and 1 mM EDTA in 100 mM Tris-HCl, pH 7.8, was preincubated for 10 min at RT, so that the GAPC2-substrate 1,3-bisphosphoglycerate was formed, and the mixture was degassed afterwards. The reaction mix was measured at RT in a quartz cuvette at 340 nm for 1–2 min before 15 nM pretreated GAPC2 was added. The spectrophotometric measurement was continued for further 5 min (Cary 3500 UV-Vis, Agilent, Santa Clara, CA, USA). Enzyme activity was calculated using the molar extinction coefficient of NADH (ε = 6220 M−1.cm−1).
The reaction mixture containing 4 mM ATP, 8 mM 3-phosphoglyceric acid disodium salt (3-PGA), 0.294 mM NADH, 90 nM phosphoglycerate kinase (PGK), 8 mM MgSO4 and 1 mM EDTA in 100 mM Tris-HCl, pH 7.8, was preincubated for 10 min at RT, so that the GAPC2-substrate 1,3-bisphosphoglycerate was formed, and the mixture was degassed afterwards. The reaction mix was measured at RT in a quartz cuvette at 340 nm for 1–2 min before 15 nM pretreated GAPC2 was added. The spectrophotometric measurement was continued for further 5 min (Cary 3500 UV-Vis, Agilent, Santa Clara, CA, USA). Enzyme activity was calculated using the molar extinction coefficient of NADH (ε = 6220 M−1.cm−1).
6
Catalytic Hydrogenation of NAD+ to NADH
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NAD+ solution (3 mM) was prepared in a phosphate buffer at pH 8.5. Both glass reaction vials containing 4 ml NAD+ solution (no catalyst) and vials containing 4 ml NAD+ solution and nickel (Alfa Aesar) and iron powder (Alfa Aesar) as solid phase catalysts, added as 26 mg Fe plus 28 mg Ni powder per ml solution, were placed in a stainless-steel reactor (Berghof). The vials were closed with PTFE septum lids which were penetrated with syringe needles (Sterican) to ensure the reaction gas could enter the vials. The closed reactor was pressurized with 5 bar of hydrogen gas and heated up to 40°C for a total of 4 h. After depressurizing the reactor, samples were transferred to 2 ml Eppendorf tubes, centrifuged for 15 min at 13,000 rpm (Biofuge fresco, Heraeus) and the supernatant was collected to spectrophotometrically observe NADH synthesis (characteristic maximum absorbance at 339 nm; Cary 3500 UV-Vis, Agilent) (see Supplementary Figure 4 ). For convenience, conversion tables relating H2 partial pressures and H2 concentrations in water at different temperatures are given in Supplementary Table 6 .
7
Characterization of Nanoparticle Properties
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The size and morphology of the NPs were analyzed using a transmission electron microscope (JEOL 2000 FX TEM) operating at 200 kV accelerating voltage. TEM samples were prepared by dropping 15 mL of NP solution on a carbon-coated copper grid (400 Mesh, Agar Scientific), where the samples were allowed to sit on the grid for at least 15 minutes before analyses. Fourier-transform infrared spectroscopy (FTIR) was carried out by drying silica NPs at 37°C for 48 hours and finally placing the dry sample onto a Cary 630 FTIR spectrometer (Agilent Technologies Ltd) for the measurement of transmittance spectra. UV-Vis absorption spectrum of ZnO and GNPs was recorded on a Cary 3500 UV-Vis (Agilent Technologies Ltd). The hydrodynamic diameter (hd) and Zeta potential (ζ) of the NPs were monitored on a Malvern Zetasizer Nano-ZS (Malvern Instruments, UK).
8
Enzymatic Assays for Lox2 and βCA1
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Fatty acid oxidation was observed spectrophotometrically at 234 nm. Preincubation of recombinant and untreated LOX2 was done in 35 mM HEPES buffer at pH 8.0 for 15 min. This included 0.5 µM of LOX2 and 0.5 µM either reduced or oxidized 2-CysPRXA. The enzymatic reactions were initiated by adding 20 µM α-linolenic acid. The β-CARBONIC ANHYDRASE 1 activity assay was used according to [19 (link),20 (link)]. Recombinant hisx6-tagged βCA1 was pretreated with 500 µM DTT for 30 min at room temperature for activation. The ßCA1 protein (50 nM) was added to 2 mL 20 mM Tris-HCl (pH 8.5 at 5°C) with 15 µg/mL phenolred (T127.2, Carl Roth, Karlsruhe, D) and 250 nM of the different 2-CysPRX variants (WT, C54D and C54S) with a final concentration of 50 nM. Then, 1.33 mL ice cold CO2-saturated water, including 15 µg/mL phenolred, was added to the cuvette to start the reaction at 2 °C. The reaction was monitored spectrophotometrically at 558 nm as decrease of absorbance of phenolred (Cary 3500 UV-Vis, Agilent). The Wilbur–Anderson Unit [19 (link),21 ] was determined as the time span required for the solution pH to drop from 8.3 to 6.3.
9
Oxidation-Reduction Modulates Cyp20-3 PPIase Activity
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Cyp20-3 protein was either oxidized by overnight dialysis at 4 °C twice in 1000-fold volume of 50 mM Na-Pi, pH 8.0, containing 10 mM H2O2 or reduced using an immobilized reductant column (Thermo-Scientific). The catalytic peptidyl-prolyl-cis/trans-isomerase (PPIase) efficiency expressed as Kcat/Km was measured as in [32 (link)] using a Cary 3500 UV–Vis spectrophotometer (Cary 3500 UV-Vis, Agilent, Santa Clara, CA, USA) and a 2.5 mL polystyrene cuvette. 100 mM of substrate (N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide) was mixed with varying amounts of pre-oxidized or reduced Cyp20-3 (5, 10, 25, 50, 75, 100 nM) in 35 mM HEPES, pH 8.0. After adding 26 µM of 12-OPDA, the sample was incubated at 8 °C and 800 rpm for 10 min. The control solution lacked Cyp20-3. The baseline was recorded at 390 nm for 3 min before the reaction was initiated by adding 0.8 mg α-chymotrypsin. Catalytic efficiencies of Cyp20-3 C54S and C171S could not be measured because of protein precipitation at the required concentration of 4 µg µL−1 in the activity test.
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