Samples for the EPR monitoring of H/D exchange on the glycyl radical were prepared by exchanging the solvent via passage over PD-10 gel filtration columns (1.5 x 5 cm, GE healthcare). These were made anoxic by washing with water containing 5 mM dithionite, then equilibrated with either anoxic water or D2O. After applying 0.5 ml of cell extract, the columns were eluted with either anoxic water or D2O, resulting in an initial exchange of water to D2O and a second exchange back to water. The recovered protein-containing eluates did not show significant dilution. The exchange of protium to deuterium was also confirmed by mixing the cell-free extract in a 1:1 ratio with anoxic D2O, using non-treated cell extract as a control sample. Extracts were transferred into EPR tubes under anaerobic conditions, secured with a clamped rubber tube, and gently frozen in liquid nitrogen. The samples were stored in liquid nitrogen until measured.