ESR MEASUREMENTS OF OXYGEN CONSUMPTION & SPIN LABEL REDUCTION

氮氧化物自旋标记和探针被应用于测量 ESR测定细胞氧化还原活性和氧浓度。当前 实验方法通常需要大量的细胞和 只监测ESR信号的幅度。这可能会导致 实验人工制品和数据曲解。一种改进的 描述了准确测量氮氧化物强度的方法。 和Line Width，这又提供了关于细胞氧化还原的信息 活动和氧气浓度。该方法是基于拟合的 基于快速卷积最小二乘算法的ESR波谱分析 适用于不均匀加宽的电渣线。所有配件 参数，包括线宽的洛伦兹分量和 从实验中提取了双积分光强 直接进行光谱分析。实验是在CELL上进行的 5×106个细胞毫升-1或更少的浓度以防止细胞拥挤 并在初始氮氧化物浓度为1 mM或100M时确保 细胞对氮氧化物的代谢不受扩散限制。 例如，用培养的小仓鼠肾脏和 腺病毒介导的含氮自由基转化仓鼠细胞 坦普尔(2，2，6，6-四甲基哌啶-N-氧基-4-01)和15N-标记 全氚代泰普酮(15NPDT) (2，2，6，6-四甲基哌啶-N-氧基-4-酮)。在这些下面 实验条件下，推导出氮氧化物的代谢率 从氮氧化物强度的变化来看，与氧气无关 集中精神。

Nitroxide spin labels and probes were applied to measurements of cell redox activities and oxygen concentration by ESR. Current experimental methods often require large numbers of cells and monitoring of only the amplitude of the ESR signal. This can result in experimental artifacts and data misinterpretation. An improved method is described for accurate measurement of nitroxide intensity and linewidth, which in turn provides information about cell redox activities and oxygen concentration. The method is based on fitting of ESR spectra using a fast convolution least-squares algorithm suitable for inhomogeneously broadened ESR lines. All fitting parameters, including the Lorentzian component of the linewidth and the double integrated intensity, are extracted from the experimental spectra directly. The experiments were carried out at cell concentrations of 5 x 106 cell ml-1 or less to prevent cell crowding and at initial nitroxide concentration of 1 mM or 100 M to ensure that nitroxide metabolism by the cells was not diffusion limited. Examples include experiments with cultured baby hamster kidney and adenovirus transformed hamster cells using the nitroxide radicals TEMPOL (2,2,6,6-tetramethylpiperidine-N-oxyl-4-0l) and 15N-labeled perdeuteriated TEMPONE (15NPDT) (2,2,6,6-tetramethylpiperidine-N-oxyl-4-one). Under these experimental conditions, the rates of nitroxide metabolism derived from changes in nitroxide intensity are independent of oxygen concentration.