Detection of the kinetics of biochemical reactions with oxygen using exchange broadening in the ESR spectra of nitroxide radicals

Backer, Joseph M.; Budker, Vladimir G.; Eremenko, S.I.; Molin, Yu. N.

doi:10.1016/0005-2728(77)90161-x

Cited by 96 publications

(71 citation statements)

References 2 publications

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“…1, are described not only to illustrate features of the instrument but also because this is a widely used compound for spin-label oximetry. In an early paper, Backer et al [19] used the resolution of the superhyperfine structure of compound II (4-carboxy-2.2.5.5-tetramethyl-3-imidazoline-3-oxide-1-oxyl) of Fig. 1 as an indicator of bimolecular collisions with dissolved O 2 .…”

Section: Spin-label Oximetrymentioning

confidence: 99%

Saturation recovery EPR and ELDOR at W-band for spin labels

Froncisz

Camenisch

Ratke

et al. 2008

Journal of Magnetic Resonance

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A reference-arm W-band (94 GHz) microwave bridge with two sample-irradiation arms for saturation recovery (SR) EPR and ELDOR experiments is described. Frequencies in each arm are derived from 2 GHz synthesizers that have a common time-base and are translated to 94 GHz in steps of 33 and 59 GHz. Intended applications are to nitroxide radical spin labels and spin probes in the liquid phase. An enabling technology is the use of a W-band loop-gap resonator (LGR) (J.W. Sidabras et al., Rev. Sci. Instrum. 78 (2007) 034701). The high efficiency parameter (8.2 GW −1/2 with sample) permits the saturating pump pulse level to be just 5 mW or less. Applications of SR EPR and ELDOR to the hydrophilic spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPONE) are described in detail. In the SR ELDOR experiment, nitrogen nuclear relaxation as well as Heisenberg exchange transfer saturation from pumped to observed hyperfine transitions. SR ELDOR was found to be an essential method for measurements of saturation transfer rates for small molecules such as TEMPONE.

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Section: Spin-label Oximetrymentioning

confidence: 99%

Saturation recovery EPR and ELDOR at W-band for spin labels

Froncisz

Camenisch

Ratke

et al. 2008

Journal of Magnetic Resonance

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“…An advantage of EPR oximetry is that it is based on pure physical interaction, Heisenberg spin exchange, between paramagnetic molecules of probe and oxygen, and does not interfere with oxygen metabolism, therefore providing a basis for noninvasive oxygen measurements in biological systems. Initially, the paramagnetic probes for EPR oximetry were almost exclusively nitroxides (Backer et al, 1977;Lai et al, 1982;Froncisz et al, 1985;Bacic et al, 1989;Chan et al, 1989;Hyde & Subszynski, 1989). Both the longitudinal (T1) and transverse (T2) relaxation times of the NRs can be affected by collisions of the nitroxide with dissolved oxygen.…”

Section: Epr-based Oximetric Applications Of the Nitroxidesmentioning

confidence: 99%

“…The first observation of oxygen-induced NR broadening in various solvents was reported by Povich (Povich, 1975). Two years later the T2 oximetry method was proposed and applied to follow mitochondrial respiration in samples containing about 100 liver cells (Backer et al, 1977). T1-sensitive EPR oximetry was developed by Hyde et al (Froncisz et al, 1985;Hyde et al, 1990).…”

Section: Epr-based Oximetric Applications Of the Nitroxidesmentioning

confidence: 99%

“…Alternatively, measurement of "the depth of resolution" of the NRs superhyperfine structure (e.g. parameter a/A for the CTPO shown in the Fig.1) provides similar or even better sensitivity to oxygen than perdeuterated NRs, and has been used for oxygen measurements in cellular and enzymatic systems (Backer et al, 1977;Sarna et al, 1980;Lai et al, 1982). However, both approaches still suffer from concentration-induced line broadening which is difficult to disentangle from oxygen-induced broadening.…”

Section: Epr-based Oximetric Applications Of the Nitroxidesmentioning

confidence: 99%

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In vivo Spectroscopy and Imaging of Nitroxide Probes

Khramtsov¹

2012

Nitroxides - Theory, Experiment and Applications

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“…With a suitable spin probe, this property can be used to quantitate 02 concentrations in solution (8)(9)(10).…”

mentioning

confidence: 99%

ESR studies of O2 uptake by Chinese hamster ovary cells during the cell cycle.

Lai¹,

Hopwood²,

Hyde³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

110

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Magnetic interactions between dissolved oxygen and nitroxide radical spin probes lead to broadening of the ESR lines. We have used a closed-chamber method based on this property to determine the maximum rate of 02 uptake per cell (Vm. per cell) in cultured mammalian cells. A suitable spin probe and a cell suspension are mixed in an aerated medium, and the rate of disappearance of dissolved 02 is measured. The effects of temperature, pH, and microwave power on the determination of dissolved oxygen in solution were studied. For Most mammalian cells are aerobic, and dissolved oxygen is vital for their growth. Elucidation of the mechanism of 02 uptake during the cell cycle is probably fundamental to the complete understanding of cell growth and cell division.There have been several reports on the measurement of 02 uptake during the cell cycle (1-4). Most previous research has been done on dividing egg cells by using the Cartesian diver technique (1) and on yeast cells by using the Clark electrode (2). To our knowledge, only two reports have appeared concerning 02 uptake by cultured mammalian cells during the cell cycle (3,4). One of the obstacles is that the existing techniques for measurement of 02 uptake by cultured mammalian cells require large amounts of.cells for each measurement, making it difficult to study mitotically synchronized cells. Most chemical means of synchronization induce multiple biochemical perturbations that obscure the interpretations of any 02 uptake measurements.It is well known that the interaction ofdissolved oxygen molecules and nitroxide free radicals through Heisenberg spin-exchange causes broadening of the ESR lines (5-7). With a suitable spin probe, this property can be used to quantitate 02 concentrations in solution (8)(9)(10).In this report, we describe an ESR closed-chamber method for determining the 02 uptake by cultured mammalian cells.Basically, a spin probe and a cell suspension are mixed in an aerated medium and the rate of disappearance of 02 is measured. Because a relatively small number of cells is required for each measurement, it has been possible in the present work to study oxygen uptake of mitotically synchronized populations of Chinese hamster ovary (CHO) cells. MATERIALS AND METHODSCell Lines. CHO cells maintained in monolayer culture from frozen stocks for several years in this laboratory were used because they are well characterized. The procedures for culturing CHO cells, either in monolayer or in suspension, and for synchronization have been described (11). The mitotic index ofcells in mitosis was about 97% and did not change appreciably during the ESR measurement.ESR Closed-Chamber Method. A mixture of 10 ml of phosphate-buffered saline (Oxoid; Dulbecco's solution A without Mg2+ and Ca2+, pH 7.4) containing 0.114 mM spin probe [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (CTPO); Aldrich] and 0.2% methyl cellulose E4M (Dow) or 0.1% agar (buffer A) was equilibrated with air at 370C with stirring for at least 10 min prior to use. About 5-10 X...

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Detection of the kinetics of biochemical reactions with oxygen using exchange broadening in the ESR spectra of nitroxide radicals

Cited by 96 publications

References 2 publications

Saturation recovery EPR and ELDOR at W-band for spin labels

Saturation recovery EPR and ELDOR at W-band for spin labels

In vivo Spectroscopy and Imaging of Nitroxide Probes

ESR studies of O2 uptake by Chinese hamster ovary cells during the cell cycle.

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