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

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 ΔB p−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 ΔB p−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.

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase dependent

“…3 and 4), our study using MD simulations carried out for single component bilayer membranes leads to the conclusion that the location of the TEMPO moiety of TEMPO-PC in bilayers is membrane phase-dependent, which we hope constructively addresses some of the issues discussed in the field. 15,17,18 Lastly, given that membrane phase or membrane fluidity depends on a number of factors, such as temperature, types of fatty acids, presence of cholesterol, membrane curvature, medium osmolality, solutes in aqueous phase, and whether the measurement is made on single or multi-bilayer vesicle, 4,[34][35][36][37][38][39][40] extra attention should be paid to the physicochemical condition of bilayers when interpreting the data collected from TEMPO-PC.…”

“…TEMPO is a six-membered cyclic nitroxide radical shielded by four methyl groups. Tethered at various positions along the hydrocarbon tail in the form of n-doxyl PC lipids, fluorophores or nitroxide spin labels including TEMPO have been employed to measure the penetration depth of solvent molecules and the flexibility of lipid tail by analyzing homogeneous line broadening of EPR spectra [1][2][3][4] and fluorescence quenching. 5 Meanwhile, TEMPO-PC, a molecular construct in which TEMPO molecule is tethered to the choline group of PC, has been used to probe the water/lipid interfacial molecular environment [6][7][8] with an estimate based on molecular model that TEMPO is positioned at 5 Å above the bilayer interface.…”

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase-dependent

“…TEMPO is a six-membered cyclic nitroxide radical shielded by four methyl groups. Tethered at various positions along the hydrocarbon tail in the form of n-doxyl PC lipids, fluorophores or nitroxide spin labels including TEMPO have been employed to measure the penetration depth of solvent molecules and the flexibility of lipid tail by analyzing homogeneous line broadening of EPR spectra [1][2][3][4] and fluorescence quenching. 5 Meanwhile, TEMPO-PC, a molecular construct in which TEMPO molecule is tethered to the choline group of PC, has been used to probe the water/lipid interfacial molecular environment [6][7][8] with an estimate based on molecular model that TEMPO is positioned at 5 Å above the bilayer interface.…”

pH of Water Intercalated into Graphite Oxide as Determined by EPR Spectroscopy

“…pH-sensitive spin probe method is a powerful tool for pH-measurements in systems which cannot be analyzed using macroscopic pH measuring equipment [24], [25], [26], [27]. This technique is extensively used for study of polymers [28] [29], biological membranes and proteins [30], [31], in vivo objects [32], [33], nanomaterials [34], [35] etc. In works [25], [32], [33],[36] pH-sensitive nitroxides were used for 3D-mapping of biological tissues and tumors.…”