Effect of Solution Ionic Strength on the pK of the Nitroxide pH EPR Probe 2,2,3,4,5,5-Hexamethylimidazolidin-1-oxyl.

Spin probe and spin labeling Electron Paramagnetic Resonance methods are indispensable research tools for solving a wide range of bioanalytical problems-from measuring microviscosity and polarity of phase-separated liquids to oxygen concentrations in tissues. One of the emerging uses of spin probes are the studies of proton transfer-related and surface electrostatic phenomena. The latter Electron Paramagnetic Resonance methods rely on molecular probes containing an additional functionality capable of reversible ionization (protonation, in particular) in the immediate proximity to an Electron Paramagnetic Resonance-active reporter group, such as (N-O) for nitroxides. The consequent formation of protonated and nonprotonated nitroxide species with different magnetic parameters (A , g ) could be readily distinguished by Electron Paramagnetic Resonance. Bioanalytical Electron Paramagnetic Resonance studies employing pH-sensitive paramagnetic probes typically involve determination of the equilibrium constant (pK ) between the protonated and nonprotonated forms of the nitroxide. However, any chemical equilibrium involving charged species, such as ionization of acids and bases, and so the reversible protonation of the nitroxide, is known to be affected by an ionic strength of the solution. Currently, only scarce data for the effect of the solution ionic strength on the experimental pK 's of the ionizable nitroxides can be found in the literature. Here we have carried out a series of Electron Paramagnetic Resonance titration experiments for aqueous solutions of 2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl (HMI) nitroxide known for one of the largest differences in the isotropic nitrogen hyperfine coupling constant A between the protonated and nonprotonated forms. Electrolyte concentration was varied over an exceptionally large range (i.e., from 0.05 to 5.0 M) to elucidate the effect of ionic strength on the ionization constant of this pH-sensitive Electron Paramagnetic Resonance probe and the data were compared to the Debye-Hückel limiting law. Effects of the ionic strength on the magnetic parameters of the ionizable nitroxides are also discussed.