title: Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes.
abstract: Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis-Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.
authors.affiliation: University of Konstanz, Departments of Chemistry and Biology, Konstanz Research School Chemical Biology (KoRS-CB), Zukunftskolleg, 78457 Konstanz, Germany. mykhailo.azarkh@uni-konstanz.de
authors.forename: Mykhailo
authors.initials: M
authors.lastname: Azarkh
authors.forename: Oliver
authors.initials: O
authors.lastname: Okle
authors.forename: Philipp
authors.initials: P
authors.lastname: Eyring
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Dietrich
authors.forename: Malte
authors.initials: M
authors.lastname: Drescher
mesh_terms: D000818:Animals; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D007202:Indicators and Reagents; D007700:Kinetics; D009589:Nitrogen Oxides / Q000737:chemistry*; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D013113:Spin Labels; D014982:Xenopus laevis
chemical_list: D003497:Cyclic N-Oxides; D007202:Indicators and Reagents; D009589:Nitrogen Oxides; D013113:Spin Labels; C037109:2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylic acid