Emxplus epr spectrometer

Superoxide formation was measured using electron paramagnetic resonance (EPR) spin trapping spectroscopy [30 (link)]. Briefly, harvested lung, liver, and spleen tissues were homogenized with 10 μg/mL chelex-treated PBS containing aprotinin, 0.5 μg/mL leupeptin, 0.7 μg/mL pepstatin, and 500 μM PMSF. The protein samples (30 μg) were mixed with 1 mM 1-hydroxy-3-carboxypyrrolidine (CPH) and 0.1 mM diethyl-tetrapentaacetic acid (DTPA) to chelate the ions of transition metals. The mixture was loaded in 50-μL glass capillary tubes (Wilmad Glass, Buena, NJ, USA). The electron paramagnetic resonance spectra were recorded using an EMX Plus EPR spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with an EMX-m40X microwave bridge operating at 9.88 GHz.

All PhoX^MED193 EPR samples were prepared in activity buffer (pH 8.0). Two hundred fifty–microliter samples of purified PhoX^MED193 at 190 μM were prepared with additives at final concentrations of either 5 mM dithionite, 20 mM dithionite, 50 mM EDTA, 1 mM PE, or no additives (no additive control). PhoX^MED193 activity buffer was used as a blank. All PhoX^MED193 samples were measured on a Bruker EMXplus EPR spectrometer equipped with a Bruker ER 4112SHQ X-band resonator at 10 K. Sample cooling was achieved using a Bruker “Stinger” cryogen free system mated to an Oxford Instruments ESR900 cryostat, and temperature control was maintained using an Oxford Instruments MercuryITC, as reported previously (75 (link)–77 (link)). The EPR spectra were measured with a microwave power of 20 dB (2.2 mW), a modulation amplitude of 5 G, a time constant of 82 ms, a conversion time of 12 ms, a sweep time of 120 s, a receiver gain of 30 dB, and an average microwave frequency of 9.385 GHz, as previously described (29 (link)). Each spectrum was averaged over four to six scans to get a better signal to noise ratio. The analysis of the continuous wave EPR spectra was performed using EasySpin toolbox (5.2.28) for the MATLAB program package (78 (link)).

All EPR samples were prepared in Tris-HCl buffer pH 7.5. Samples containing ~300 μM of wild-type TtCBD and TtCBD-H132A were transferred into 4 mm outer diameter/3 mm inner diameter Suprasil quartz EPR tubes (Wilmad LabGlass) and frozen in liquid N₂. The photoactivation of the TtCBD and TtCBD-H132A was carried out at room temperature under anaerobic conditions. Optical irradiation at 530 nm (500 mW) was accomplished (for the specified duration described in the text/legend) using a Thorlabs Mounted High Power LED (M530L3) with the output beam collimated using a Thorlabs collimation adaptor (SM2F32-A). All EPR samples were measured on a Bruker EMXplus EPR spectrometer equipped with a Bruker ER 4112SHQ X-band resonator. Sample cooling was achieved using a Bruker Stinger^{34 (link)} cryogen-free system mated to an Oxford Instruments ESR900 cryostat, and temperature was controlled using an Oxford Instruments MercuryITC. The optimum conditions used for recording the spectra are given below; microwave power 30 dB (0.22 mW), modulation amplitude 5 G, time constant 82 ms, conversion time 25 ms, sweep time 90 s, receiver gain 30 dB and an average microwave frequency of 9.383 GHz. All EPR spectra were measured as a frozen solution at 20 K, respectively. The analysis of the continuous wave EPR spectra were performed using EasySpin toolbox (5.2.28) for the Matlab program package^{35 (link)}.

For the Fenton reaction (Fe²⁺ + H₂O₂ → Fe³⁺ + ⋅OH + OH⁻), 80 µl of reaction mixture containing 250 µM ferrous sulphate, 250 µM H₂O₂, 80 mM spin trap DMPO, and 1 mM of 1,4 DHP was transferred to a micro pipettes tube for measurement of the electron spin resonance (ESR) spectra of DMPO-OH radicals. ESR spectra of the spin trap and radical complex were recorded at room temperature using an EMX-plus EPR spectrometer (Bruker, Germany). The EPR instrumental settings for field scan were as follows: field sweep—100G; microwave frequency—9.84 GHz; microwave power—15.9 mW; modulation amplitude—1 G; conversion time—163 ms; time constant—327 ms; sweep time –83 s; receiver gain–1⋅10⁴; resolution—512 points for 1 scan.

The EPR spectra of Cu-containing zeolites were recorded as the first derivative of the absorption signal of a EMXplus EPR spectrometer (Bruker, Karlsruhe, Germany) in the X-band (9.4 GHz). A variable temperature unit ER4141VTM was used for temperature variation. The EPR spectra were simulated by the program SimFonia (Bruker) and the quantitative analysis was performed by the licensed program SpinCount (Bruker).