Glutathione

Typical uses are oxidative-stress models with GSH depletion as the readout, glutathione peroxidase and glutathione S-transferase assays, and GSH:GSSG measurements. Work on GSH synthesis looks at glutamate cysteine ligase and its regulation by Nrf2 through the antioxidant response element, AP-1 and NF-κB (Lu, 2013).

GSH is the reduced form, with a free thiol on its cysteine. GSSG is the disulfide formed when two GSH molecules are oxidised together. Glutathione reductase converts GSSG back to GSH using NADPH. The GSH:GSSG ratio is one of the most widely measured redox parameters in cell biology. Dissolved GSH oxidises in air, so solutions for ratio work need protecting from oxygen.

Glutathione (gamma-L-glutamyl-L-cysteinyl-glycine; GSH) is the principal low-molecular-weight thiol antioxidant of mammalian cells, with the molecular formula C₁₀H₁₇N₃O₆S, a molecular weight of 307.32 g/mol and CAS number 70-18-8. It is a tripeptide in which cysteine is flanked by glutamate and glycine, and, unusually, glutamate is joined to cysteine through its gamma-carboxyl group rather than the alpha-carboxyl used in an ordinary peptide bond. That gamma-glutamyl linkage is functionally important: it makes GSH resistant to the peptidases that would otherwise degrade a normal tripeptide, so intracellular GSH is cleaved only by gamma-glutamyl transpeptidase on the extracellular face of the plasma membrane. Biosynthesis is a two-step, ATP-dependent pathway (Meister and Anderson, Annual Review of Biochemistry, 1983): glutamate-cysteine ligase condenses glutamate and cysteine to gamma-glutamylcysteine, and glutathione synthetase then adds glycine. Because the first enzyme is both feedback-inhibited by GSH and limited by cysteine supply, cysteine availability is the dominant control point for synthesis (Lu, Biochimica et Biophysica Acta, 2013). Glutathione functions as a cellular redox buffer: it cycles between the reduced thiol form (GSH) and the oxidised disulfide (GSSG) through NADPH-dependent glutathione reductase, and healthy cells maintain a GSH:GSSG ratio of roughly 100:1. It acts as a direct radical scavenger, as the obligate electron donor for the glutathione peroxidases that reduce hydrogen peroxide and lipid peroxides, and as a substrate for the glutathione S-transferases that conjugate electrophiles and xenobiotics for export (Forman, Zhang and Rinna, Molecular Aspects of Medicine, 2009). Sies (Free Radical Biology and Medicine, 1999) framed these combined roles as a general cellular defence system against oxidative challenge, and the same review literature documents a well-replicated decline in tissue GSH with age. For experimental work the key practical point is that reduced GSH is air-oxidisable: dissolved GSH dimerises to GSSG on exposure to oxygen, so any assay of the reduced form must protect the thiols, by acid precipitation, alkylation or a reducing environment, if the GSH:GSSG couple is the measurement of interest.