Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

Abstract Due to fast relaxation processes of transition metal ions, electron paramagnetic resonance (EPR) spectroscopy metalloproteins needs be performed at cryogenic temperatures. To avoid damaging the biological system upon freezing, a cryoprotectant is generally added sample as glassing agent. Even though cryoprotectants are expected inert substances, evidences in literature show their non-innocent role altering shape EPR spectra proteins and objects general. In this work we conduct systematic study on impact several experimental factors—such buffer composition, choice cryoprotectant, pH temperature—on spectrum myoglobin, taken reference for being well-characterized heme-containing protein. We focus high-pH buffers induce investigate alkaline ferric myoglobin (pKa ~ 8.9). A combined approach continuous-wave UV–visible absorption shows that using particular pairs determines considerable variation effect enhanced temperature. addition, phase memory times were measured evaluate efficiency different compared with spectral linewidths EPR. Our findings suggest among selected ethylene glycol rather effective, even more than widely used glycerol, without having unwanted effects.