Abstract

EPR studies of cellular oxygen and nitroxide metabolism often involve a variety of experimental conditions including different sample preparations, cellular concentrations, and spectrometer settings. Post-processing of the resulting spectra also varies greatly. Here, we show that experimental conditions can affect results often in unexpected ways. We present data on the effects of cell density, type of cell bathing media, and nitroxide concentration in the sample, and power levels and modulation intensity setting of the spectrometer, on oxygen and nitroxide consumption results. We also compare information obtained from simple line height measurements with information obtained from least-squares spectral simulation techniques. Our most consistent results are obtained at low (105 cells/ml or less) cell density, media which alone does not reduce nitroxides, low (0.1mW) power, modulation levels appropriate for good (100:1 or better) signal-to-noise, and lineshape analysis by a fast convolution algorithm combined with Levenberg-Marquard optimization (for determining oxygen and nitroxide concentrations).

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001811-13
Application #
6120708
Study Section
Project Start
1998-04-15
Project End
1999-11-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
13
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
University of Illinois at Chicago
Department
Type
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612

  Publications

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Denisov, Ilia G; Makris, Thomas M; Sligar, Stephen G (2002) Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis. J Biol Chem 277:42706-10
Denisov, I G; Hung, S C; Weiss, K E et al. (2001) Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119. J Inorg Biochem 87:215-26
Atsarkin, V A; Demidov, V V; Vasneva, G A et al. (2001) Mechanism of oxygen response in carbon-based sensors. J Magn Reson 149:85-9
Mangels, M L; Harper, A C; Smirnov, A I et al. (2001) Investigating magnetically aligned phospholipid bilayers with EPR spectroscopy at 94 GHz. J Magn Reson 151:253-9
Breitzer, J G; Smirnov, A I; Szczepura, L F et al. (2001) Redox properties of C6S8(n-) and C3S5(n-) (n = 0, 1, 2): stable radicals and unusual structural properties for C-S-S-C bonds. Inorg Chem 40:1421-9
Kirkor, E S; Scheeline, A (2000) Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator. Eur J Biochem 267:5014-22
Rapoport, N; Smirnov, A I; Pitt, W G et al. (1999) Bioreduction of Tempone and spin-labeled gentamicin by gram-negative bacteria: kinetics and effect of ultrasound. Arch Biochem Biophys 362:233-41
Maringanti, S; Imlay, J A (1999) An intracellular iron chelator pleiotropically suppresses enzymatic and growth defects of superoxide dismutase-deficient Escherichia coli. J Bacteriol 181:3792-802

Showing the most recent 10 out of 16 publications