Takeshita Keizo, Saito Keita, Ueda Jun-ichi, Anzai Kazunori, Ozawa Toshihiko
Redox Regulation Research Group, National Institute of Radiological Sciences, 9-1 Anagawa-4, Inage-ku, Chiba 263-8555, Japan.
Biochim Biophys Acta. 2002 Nov 14;1573(2):156-64. doi: 10.1016/s0304-4165(02)00420-8.
The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.
研究了硝酰自旋探针的化学结构对在活性氧（ROS）存在下电子自旋共振（ESR）信号丧失速率的影响。当自旋探针在半胱氨酸或烟酰胺腺嘌呤二核苷酸（NADH）存在下与羟基自由基（·OH）或超氧阴离子自由基（O₂⁻·）反应时，探针ESR信号的丧失取决于其环结构和取代基。吡咯烷硝酰探针相对抵抗由半胱氨酸/NADH存在下的O₂⁻·引起的信号衰减。这些反应的信号衰减速率与报道的自旋探针的硝酰/氧鎓铵偶联的氧化还原电位相关，表明两种情况下的信号衰减机制都涉及硝酰基团的氧化。分别使用甘露醇和超氧化物歧化酶（SOD）作为竞争标准，估算了在半胱氨酸存在下自旋探针与·OH之间以及自旋探针与O₂⁻·之间反应的表观速率常数。自旋探针与·OH反应的速率常数约为10⁹ M⁻¹ s⁻¹，远高于在半胱氨酸存在下探针与O₂⁻·反应的速率常数（10³ - 10⁴ M⁻¹ s⁻¹）。这些基础数据对于通过体内ESR光谱法测量活体动物中的·OH和O₂⁻·很有用。
