Emx x band epr spectrometer

TMP-V-T stock solutions were diluted to 0.1 mM final concentration, and 10-fold molar excess of K₃[Fe(SCN)₆] was added to reoxidize any reduced radical. CW-EPR data were acquired at room temperature using a Bruker EMX X-band EPR spectrometer at a microwave frequency of 9.756 GHz, with a center field of 3480 G, a sweep width of 100 G, a modulation frequency of 100 kHz, a modulation amplitude of 1.00 G, a conversion time of 164 s, and a microwave power of 20.1 mW. The integrated intensity of the reoxidized sample was compared to that of a control without added K₃[Fe(SCN)₆] to determine the reduced population.

EPR spectra are acquired using a Bruker EMX X-band EPR spectrometer with ER041X microwave bridge, and EMX080 magnet power supply and dipole electromagnet (10-inch diameter pole face). Magnetic field modulation (100 kHz frequency) and signal acquisition were controlled by connecting the modulation reference out from the EMX spectrometer to a Stanford Research Systems model SR810 DSP lock-in amplifier (time constant 6dB [3 ms], 500 mV sensitivity, low noise mode). Field modulation amplitude was regulated by amplifying the 100 kHz signal output from the lock-in amplifier through a custom-built amplifier, providing a 0.5 mT modulation amplitude within the SRA housing. A slide-screw tuner was used to adjust the coupling of the resonator and microwave bridge. Spectral acquisition parameters were the following: 15.0 mT sweep width, 5.12 s scan time, 10.49 ms time constant, 5.12 ms conversion time, 10.01 output microwave power, receiver gain 5×10⁴, and averages of 25 or 50, 7 s (5.12 s field scan time plus 1.88 s system reset and equilibrium time) scans.

Samples prepared for EPR spectroscopy were prepared by two protocols (C) and (D). In protocol C, wt β₂ was mixed with CDP and ATP, and reaction was initiated with addition of C₄₃₉(U/S) α₂ to a final concentration of 50 μM α₂β₂, 1 mM CDP and 3 mM ATP in assay buffer in a total volume of 220 μL and then transferred to a Wilmad 4 mm precision quartz EPR tube, all under an inert atmosphere in a glove box at 4 °C. The reaction was quenched by hand following transfer (~15 s) into liquid N₂ and stored cryogenically. In protocol D, the EPR tube was initially loaded with wt β₂ (19 μL), and a premade solution of C₄₃₉(U/S) α₂, CDP and ATP in assay buffer supplemented with 20% glycerol (201 μL) was swiftly added in a single step in the EPR tube and rapidly frozen in liquid N2 cooled isopentane. This protocol reduced the quenching time to ~3 s. Samples were measured directly after freezing on a Bruker EMX X-band EPR spectrometer equipped with a recirculating helium cryostat cooled ER4199HS cavity. EPR parameters in general were as follows: microwave frequency, 9.3–9.8 GHz; modulation amplitude, 1.00 G; modulation frequency, 100 kHz; and time constant, 40.96 ms. The temperature and microwave power were varied over a wide range and individual spectra are reported with corresponding values.