131548 20240319080948.0 doi 10.1007/s00723-021-01428-w sideral 126070 ART-2022-126070 eng Serra, I Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin 2022 Access copy available to the general public Unrestricted 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 similar to 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. info:eu-repo/grantAgreement/EC/H2020/813209/EU/Paramagnetic Species in Catalysis Research. A Unified Approach Towards Heterogeneous, Homogeneous and Enzyme Catalysis/PARACAT This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 813209-PARACAT info:eu-repo/semantics/openAccess by http://creativecommons.org/licenses/by/3.0/es/ 1.0 2022 SPECTROSCOPY 32 / 41 = 0.78 2022 Q4 T3 PHYSICS, ATOMIC, MOLECULAR & CHEMICAL 32 / 35 = 0.914 2022 Q4 T3 0.234 2022 Atomic and Molecular Physics, and Optics 2022 Q4 1.7 2022 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion García Rubio, I. Universidad de Zaragoza (orcid)0000-0002-1827-1250 Van Doorslaer, S. 2003 395 Universidad de Zaragoza Dpto. Física Materia Condensa. Área Física Materia Condensada 53 (2022), 1105-1119 Appl. magn. reson. APPLIED MAGNETIC RESONANCE 0937-9347 1392992 http://zaguan.unizar.es/record/131548/files/texto_completo.pdf Versión publicada 1267218 http://zaguan.unizar.es/record/131548/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:131548 articulos driver 2024-03-18-12:45:49 ARTICLE