Nitroxide reduction experiments are usually carried out at X-band (8.8-9.5 GHz) with a relatively large number of cells (typically 107 cells in 100 ?l). Such a large number of cells is not always available for experimentation. For example, it is of interest to probe the cellular metabolism during their transformation by adenovirus when, initially, only few cells are involved. Additionally, high concentrations of cells in X-band experiments often result in inhomogeneous cell distribution in a sample tube and eventually in long diffusion paths for the metabolites and complicated apparent kinetics. We already showed (Rocky Mountain Conference, 1994) that the number of cells in the nitroxide reduction experiments can be reduced to only 900 by use of a W-band (95 GHz) EPR spectrometer developed at the Illinois EPR Research Center (IERC). By applying a resonator with a higher Q-value, careful sample positioning, and lineshape fitting software we were able to decrease this number to six cell s in a sample. We have measured reduction rate of TEMPONE (T=36.6 oC) at both X-band (3x106 cells in 30 ?l) and W-band (6 cells in 7.5 nanoliters). The rate measured from W-band experiments (3.5x10-16 molesxmin-1xcell-1) was essentially the same as that found from X-band (3.3X10-16 molesxmin-1xcell-1). From signal-to-noise considerations, we infer that it will be possible to observe nitroxide reduction by a single cell at W-band. This would open the path towards measurements of cell redox changes in which only a few cells participate. This is an ongoing study.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001811-13
Application #
6120651
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
Name
University of Illinois at Chicago
Department
Type
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Hurth, Kyle M; Nilges, Mark J; Carlson, Kathryn E et al. (2004) Ligand-induced changes in estrogen receptor conformation as measured by site-directed spin labeling. Biochemistry 43:1891-907
Woodmansee, Anh N; Imlay, James A (2002) Reduced flavins promote oxidative DNA damage in non-respiring Escherichia coli by delivering electrons to intracellular free iron. J Biol Chem 277:34055-66
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
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
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
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

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