Margita Kaleigh, Voinov Maxim A, Smirnov Alex I
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, 27606, NC, USA.
Cell Biochem Biophys. 2017 Jun;75(2):185-193. doi: 10.1007/s12013-017-0780-y. Epub 2017 Feb 17.
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.
自旋探针和自旋标记电子顺磁共振方法是解决广泛生物分析问题不可或缺的研究工具，这些问题涵盖从测量相分离液体的微粘度和极性到组织中的氧浓度等各个方面。自旋探针的新兴用途之一是研究与质子转移相关的现象和表面静电现象。后一种电子顺磁共振方法依赖于分子探针，该探针在紧邻电子顺磁共振活性报告基团（例如用于氮氧化物的（N - O））处含有能够进行可逆电离（特别是质子化）的附加功能。通过电子顺磁共振可以很容易地区分具有不同磁参数（A，g）的质子化和非质子化氮氧化物物种的形成。采用对pH敏感的顺磁探针的生物分析电子顺磁共振研究通常涉及测定氮氧化物质子化和非质子化形式之间的平衡常数（pK）。然而，任何涉及带电物种的化学平衡，例如酸和碱的电离，以及因此氮氧化物的可逆质子化，都已知会受到溶液离子强度的影响。目前，在文献中只能找到关于溶液离子强度对可电离氮氧化物实验pK'影响的稀少数据。在此，我们对2,2,3,4,5,5 - 六甲基咪唑啉 - 1 - 氧基（HMI）氮氧化物的水溶液进行了一系列电子顺磁共振滴定实验，该氮氧化物以质子化和非质子化形式之间的各向同性氮超精细耦合常数A的最大差异之一而闻名。电解质浓度在非常大的范围内变化（即从0.05到5.0 M），以阐明离子强度对这种对pH敏感的电子顺磁共振探针电离常数的影响，并将数据与德拜 - 休克尔极限定律进行比较。还讨论了离子强度对可电离氮氧化物磁参数的影响。
