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.

氮氧化物自旋探针和自旋标记广泛用于 EPR 血氧饱和度研究，范围从体内研究到通过位点特异性氧可及性实验分析脂质双层膜和蛋白质的异质环境。在此类实验中，尽管已知氮氧化物部分上的自旋密度局部化受局部极性影响，但通常假定氮氧化物和分子氧之间自旋交换相互作用的有效碰撞距离相同。此外，一些生物物理学研究，例如涉及脂质双层的研究，通常使用结构不同的氮氧化合物进行分析，但在假设氮氧化合物电子弛豫时间变化与局部氧浓度和平移扩散系数的乘积之间的比例保持不变的情况下分析结果。在这里，我们通过测量空气中的分子氧和大气压下的纯氧对六种结构不同的氮氧化物（包括能够可逆质子化的氮氧化物）的连续波 EPR 光谱的影响，对这种普遍但尚未验证的假设进行了实验验证。使用卷积拟合模型分析数据，该模型通过比较在两种不同氧浓度下测量的 EPR 光谱，可以提取峰间宽度 我们通过测量空气中的分子氧和大气压下的纯氧对六种结构不同的氮氧化物（包括能够可逆质子化的氮氧化物）的连续波 EPR 光谱的影响，对这种普遍但尚未验证的假设进行了实验验证。使用卷积拟合模型分析数据，该模型通过比较在两种不同氧浓度下测量的 EPR 光谱，可以提取峰间宽度 我们通过测量空气中的分子氧和大气压下的纯氧对六种结构不同的氮氧化物（包括能够可逆质子化的氮氧化物）的连续波 EPR 光谱的影响，对这种普遍但尚未验证的假设进行了实验验证。使用卷积拟合模型分析数据，该模型通过比较在两种不同氧浓度下测量的 EPR 光谱，可以提取峰间宽度\({\Delta }B_{{{\text{p}} - {\text{p}}}}\)是表征氧展宽效应的洛伦兹函数。虽然通过比较用氮气和纯氧平衡的氮氧化物溶液的 EPR 光谱测量的氧增宽数据显示出明显的趋势，这是由于氮氧化物结构的差异而合理化的，但\({\Delta }B_{{{\text{ p}} - {\text{p}}}}\)对于各种氮氧化物属于约。± 5%。当电解质以生理浓度存在时，这种变化与水溶液中分子氧的“盐析”效应相当。总体而言，这些数据进一步确立了氮氧化物作为强大的 EPR 分子探针，可在各种条件（如 pH 值和电解质浓度）下测量局部氧浓度及其平移扩散系数的乘积。