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EPR Oximetry with Nitroxides: Effects of Molecular Structure, pH, and Electrolyte Concentration

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Abstract

Nitroxide spin probes and spin labels are broadly utilized in EPR oximetry studies ranging from in vivo investigation to profiling heterogeneous environment of lipid bilayer membranes and proteins by site-specific oxygen-accessibility experiments. In such experiments, effective collision distances of the spin exchange interaction between the nitroxide and molecular oxygen are typically assumed to be the same even though localization of spin density over the nitroxide moiety is known to be affected by local polarity. Furthermore, some biophysical studies, such as those involving lipid bilayers, are often carried out with structurally different nitroxides but the results are analyzed under an assumption that proportionality between changes in the nitroxide electronic relaxation times and the product of the local oxygen concentration and translational diffusion coefficients remain the same. Here, we present an experimental verification of such a prevailing but not yet verified assumption by measuring effects of molecular oxygen in air and pure oxygen at atmospheric pressure on continuous wave EPR spectra of six structurally different nitroxides including those capable of reversible protonation. The data were analyzed using a convolution fitting model, which, by comparing EPR spectra measured at two different oxygen concentrations, allows one to extract the peak-to-peak width \({\Delta }B_{{{\text{p}} - {\text{p}}}}\) of a Lorentzian function that characterizes the effect of oxygen broadening. While the data on oxygen broadening measured by comparing EPR spectra of nitroxide solutions equilibrated with nitrogen and pure oxygen show clear trends that are rationalized by differences in the nitroxide structure, the overall variations in \({\Delta }B_{{{\text{p}} - {\text{p}}}}\) for various nitroxides fall within ca. ± 5%. Such variations are comparable to the effect of “salting-out” of molecular oxygen in aqueous solutions when electrolytes are present in physiological concentrations. Overall, the data further establish nitroxides as robust EPR molecular probes to measure the product of local oxygen concentration and its translational diffusion coefficient under a wide range of conditions such as pH and electrolyte concentrations.

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Acknowledgements

T.I.S. and A.I.S. are particularly thankful to Prof. H. M. Swartz who brought the subject of EPR oximetry with nitroxides to their attention back in the early 90s. This work was in part supported by U.S. DOE Contract DE-FG02-02ER15354 to A.I.S.

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  1. Department of Chemistry, North Carolina State University, Raleigh, NC, 27606-8204, USA

    Maxim A. Voinov, Tatyana I. Smirnova & Alex I. Smirnov

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  1. Maxim A. Voinov
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Voinov, M.A., Smirnova, T.I. & Smirnov, A.I. EPR Oximetry with Nitroxides: Effects of Molecular Structure, pH, and Electrolyte Concentration. Appl Magn Reson 53, 247–264 (2022). https://doi.org/10.1007/s00723-021-01446-8

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