ESR MEASUREMENTS OF OXYGEN CONSUMPTION & SPIN LABEL REDUCTION

氮氧化物自旋标记和探针应用于测量 细胞氧化还原活性和氧浓度通过ESR。 电流 实验方法通常需要大量的细胞， 仅监测ESR信号的幅度。 这可导致 实验文物和数据错误解读 一种改进 介绍了一种准确测量氮氧自由基强度的方法 和线宽，这反过来又提供了有关电池氧化还原的信息 活性和氧浓度。 该方法是基于拟合 用快速卷积最小二乘算法对ESR谱进行分析 适用于非均匀加宽的ESR谱线。 所有管接头 参数，包括线宽的洛伦兹分量和 从实验中提取了双积分强度， 光谱直接。 实验在细胞中进行。 浓度为5 x 106细胞ml-1或更低，以防止细胞拥挤 并且在初始氮氧化物浓度为1 mM或100 M确保 细胞的氮氧代谢不是扩散限制的。 例子包括用培养的幼仓鼠肾和 腺病毒转化仓鼠细胞使用氮氧自由基 TEMPOL（2，2，6，6-四甲基哌啶-N-氧基-4-01）和15 N-标记 全氘代TEMPONE（15 NPDT） （2，2，6，6-四甲基哌啶-N-氧基-4-酮）。 根据这些 实验条件下，氮氧化物代谢率推导 氮氧化物强度的变化与氧无关 浓度.

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.