In-Cell Redox Chemistry DOI: 10.1002/anie.201308004 Probing the Intracellular Glutathione Redox Potential by In-Cell NMR Spectroscopy** Steve Y. Rhieu,* Aaron A. Urbas, Daniel W. Bearden, John P. Marino, Katrice A. Lippa, and Vytas Reipa* Abstract: Non-invasive and real-time analysis of cellular redox processes has been greatly hampered by lack of suitable measurement techniques. Here we describe an in-cell nuclear magnetic resonance (NMR) based method for measuring the intracellular glutathione redox potential by direct and quanti- tative measurement of isotopically labeled glutathione intro- duced exogenously into living yeast. By using this approach, perturbations in the cellular glutathione redox homeostasis were also monitored as yeast cells were subjected to oxidative stress. Changes in intracellular redox potential are known to exert effects on gene expression that regulates major transitions (for example, proliferation, differentiation, and apoptosis) during the cell cycle. [1] Glutathione, with a cytosolic concen- tration ranging from 1 to 11 mm, [2] is the most abundant nonprotein thiol that plays an important role in modulating the intracellular redox potential. In cells, glutathione is present predominantly in the reduced form (GSH), which can be converted into the oxidized form (GSSG) during oxidative stress or detoxification of xenobiotics. Due to the abundance and reducing capacity of glutathione (standard redox potential E 0’ GSSG/2GSH = 240 mV versus the normal hydrogen electrode [3] ), the redox state of glutathione is the best indicator of intracellular redox potential. As a result, a great deal of scientific research has explored methods for measuring intracellular levels of GSH and GSSG to allow determination of the redox potential of glutathione in accordance with the Nernst equation. Most existing methods for quantifying the levels of glutathione, however, either lack a well-defined specificity or disrupt cellular integrity. In an effort to develop a method enabling nondisruptive and glutathione-specific redox meas- urements, a redox-sensitive green fluorescent protein fused with human glutaredoxin-1 (that is, Grx1-roGFP2) was recently introduced to demonstrate dynamic live imaging of the intracellular glutathione redox potential. [4] Despite the responsive glutathione redox potential readout from this method, measurement of the intracellular redox potential through direct quantification of both GSH and GSSG in a non-invasive manner has yet to be established. Herein, we demonstrate the use of nuclear magnetic resonance (NMR) spectroscopy as a tool to measure the intracellular redox potential of living cells. Since the seminal work of Dçtsch and co-workers, [5] a technique called in-cell NMR spectroscopy has been exploited to examine the structure, dynamics, and interactions of proteins in their native environment. [6–10] We utilized isotopically labeled reduced glutathione (that is, GSH-(glycine- 13 C 2 , 15 N)), denoted hereafter as GSH*, because NMR methods could then be employed to selectively observe the signal of the molecule of interest, with all of the other signals from the complex matrix of molecular species that make up the cell being filtered away. The structure of GSH* was confirmed by 1 H– 1 H total correlation spectroscopy (TOCSY) data (see the Supporting Information, Figure S1). Initially, time-course 1D 1 H– 15 N heteronuclear single quantum coherence (HSQC) spectra of GSH* dissolved in 20 mm potassium phosphate buffer at pH 7.0 were obtained over a period of 19.5 h (see the Supporting Information, Figure S2a). The amide proton of the glycine residue gave rise to two resonances, observed at d = 8.17 and 8.2 ppm in the 1 H dimension, which correspond to protons from GSH* and GSSG-(glycine- 13 C 2 , 15 N), denoted hereafter as GSSG*, respectively. It is well known that GSH is [*] Dr. S.Y. Rhieu, Dr. A.A. Urbas, Dr. V. Reipa Biosystems and Biomaterials Division National Institute of Standards and Technology Gaithersburg, MD 20899 (USA) E-mail: steve.rhieu@nist.gov vytautas.reipa@nist.gov Dr. D. W. Bearden Hollings Marine Laboratory, Chemical Sciences Division National Institute of Standards and Technology Charleston, SC 29412 (USA) Dr. J. P. Marino Institute for Bioscience and Biotechnology Research Biomolecular Measurement Division National Institute of Standards and Technology Rockville, MD 20850 (USA) Dr. K. A. Lippa Chemical Sciences Division National Institute of Standards and Technology Gaithersburg, MD 20899 (USA) [**] This research was conducted while S.Y.R. held a National Research Council Research Associateship at the National Institute of Stand- ards and Technology. We thank Dr. S. M. Da Silva for her assistance with the fluorescence microscopy and Dr. D. I. Freedberg for his comments on the manuscript. Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201308004. A ngewandte Chemi e 447 Angew. Chem. Int. Ed. 2014, 53, 447 –450  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim