Esr 900 continuous flow helium cryostat

The OcwA solution was prepared in 20 mM potassium phosphate buffer (pH 7.6) with 100 mM KCl to a final concentration of 200 μM. The reduced state was obtained by the addition of an excess of sodium borohydride. EPR spectra were recorded on a Bruker ESP 380 spectrometer equipped with an ESR 900 continuous-flow helium cryostat (Oxford Instruments). The conditions were temperature, 7 K; microwave frequency, 9.39 GHz; modulation amplitude, 1.0 mT; and microwave power, 2 mW.

EPR spectra were collected on a Bruker EMX spectrometer equipped with an ESR 900 continuous-flow helium cryostat from Oxford Instruments and were recorded at 9.39 GHz microwave frequency, 2.0 mW microwave power, 1 mT modulation amplitude, at 8 K.

The protein sample (as prepared for metal analysis, described above) was incubated with 1 mM EDTA at 4 °C for 16 h. A desalting column (Zeba Spin Desalting Columns 40K, ThermoFisher Scientific) was centrifuged at 1000g for 2 min at 4 °C to remove storage solution and washed thrice with storage buffer consisting of 100 mM NaCl, 1 mM 2-mercaptoethanol and 50 mM HEPES-KOH, pH 7.0, pre-equilibrated with 0.1% (w/v) Chelex 100 resin, Na²⁺ form. The sample was slowly applied to the center of the resin bed and eluted with 900 µl of Chelex-treated storage buffer. Metal-free aldolase was collected by centrifuging at 3000g for 2 min at 4 °C and concentrated to 35 mg/ml. The solution was dialyzed against 100 mM NaCl, 1 mM 2-mercaptoethanol, 50 mM HEPES-KOH, pH 7.0 containing 1 mM MnCl₂. EPR spectra were recorded on a Bruker ELEXSYS-II E500 spectrometer fitted with a Bruker 4116DM dual mode resonator. Sample temperature was maintained at 5 K using an Oxford Instruments ESR900 continuous flow helium cryostat. Samples (200 μl) contained 35 mg protein/ml. Instrument parameters were as follows: 9.643431 GHz, 3550 Gauss center field, 7000 field sweep range, 40 ms conversion time, 10 Gauss modulation amplitude, 100 kHz modulation frequency, 7001 pts, 28 dB microwave attenuation (0.3170 mW). Parallel-mode X-band EPR was used to test for the presence of Mn(III) at 5 K.