18] Measurement of glutathione redox state in cytosol and secretory pathway of cultured cells

Abstract

Publisher Summary The intracellular glutathione redox state has been suggested to play an important role in metabolic regulation, cellular activation, proliferation, gene expression and mRNA stability, HIV expression, and protein folding. The fact that glutathione redox states are compartmentalized in cells supports its importance in biological processes. The glutathione redox state is defined by the ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG). The glutathione redox state is defined by the ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG). These investigations necessitate accurate and reliable methodology for measuring intracellular glutathione redox states, which is hampered by thiol autoxidation and the presence of cellular enzymes capable of modifying glutathione status (GSH/GSSG ratio and their concentrations) during sample preparation and analysis. The enzymes included are glutathione peroxidase, glutathione reductase, thiol transferases, oxidases, and thioredoxin. Measurement of the cytosolic redox state is difficult as the concentration of GSSG is orders of magnitude lower than that of GSH, such that an increase or decrease in GSSG concentration during sample preparation and analysis can significantly change the glutathione redox state. In addition, the measurement of compartmentalized glutathione redox state in the endoplasmic reticulum (ER) of cells poses an even greater challenge to these investigations. This chapter discusses methodologies for measuring whole-cell and ER glutathione redox states in cultured cells.