Monitoring Glutathione in Saccharomyces cerevisiae by In-cell NMR Spectroscopy
Steve Y. Rhieu1, Aaron A. Urbas1, Katrice A. Lippa2, Daniel W. Bearden2, John P. Marino3, and Vytas Reipa1
1Biosystems and Biomaterials Division, 2Chemical Sciences Division, 3Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg MD 20899
Glutathione is the most abundant non-protein thiol in cells and exists mainly in its reduced form (GSH), which can be converted to its oxidized form (glutathione disulfide or GSSG) during oxidative stress or detoxification of xenobiotics. Owing to its abundance (cytosolic glutathione concentrations ranging from 1 to 10 mM) and reducing capacity (E0′GSSG/GSH = –240 mV vs. NHE), glutathione is considered to be the most effective redox buffer in cells. More importantly, many cellular bioprocesses are directly related to the ratio of GSH to GSSG together with the concentration of GSH determining the cytosolic redox potential in accordance with the Nernst equation. Therefore, an easy route to quantifying glutathione in biological samples is highly desirable. However, current methods capable of measuring the intracellular glutathione either often lack a well-defined specificity or disrupt cellular integrity. Here, we present the development and characterization of in-cell NMR spectroscopy-based method to monitor glutathione in the living yeast cells. Our preliminary results show that isotopically labeled glutathione (i.e., glutathione-(glycine-13C2,15N)) is successfully delivered into S. cerevisiae BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0 gsh1D::KanMX4) and can be directly observed, making in-cell NMR a promising tool to measure the glutathione in live cells.