Simultaneous ESR measurements of the kinetics of oxygen consumption and spin label reduction by mammalian cells

Abstract

AbstractNitroxide 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 × 106 cell ml−1 or less to prevent cell crowding and at initial nitroxide concentration of 1 mM or 100 μM 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‐ol) 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.