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-exper- iments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrro- lidinyl-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.