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