Glutathione

The tripeptide of glutamate, cysteine and glycine, with the glutamate joined through its side-chain carboxyl (the gamma linkage) and a free thiol on the cysteine. Drawn in the PepXtide structural library, where its computed mass matches the published molecular weight.

That gamma linkage is not a curiosity. Because the glutamate is attached through its side chain rather than its alpha-carboxyl, ordinary peptidases do not recognise the bond, and the gamma-glutamyl transferases are the only enzyme class known to cleave it. The molecule is assembled in two ATP-dependent steps, by glutamate-cysteine ligase, which is the rate-limiting enzyme, and then by glutathione synthetase.

Its biochemistry is organised around a single redox couple, reduced glutathione against the oxidised disulfide dimer. Glutathione peroxidase 1, a selenocysteine enzyme, uses two molecules of the reduced form to reduce hydrogen peroxide or a fatty-acid hydroperoxide to water or the corresponding alcohol; glutathione peroxidase 4 performs the equivalent reaction on phospholipid hydroperoxides within membranes. Glutathione reductase, an FAD enzyme, regenerates the reduced form at the expense of NADPH, and the glutathione S-transferases conjugate the thiol to electrophiles. The half-cell potential of the couple is commonly cited around -240 millivolts, though the figure depends on the cellular state being described and a single value should not be treated as a constant.

For bench work the governing number is the thiol pKa of 8.94. At pH 7.4 only about 2.8 percent of the molecule is present as the reactive thiolate, so nucleophilic reactivity rises steeply with pH; the same shift is what accelerates air oxidation in alkaline buffers.

One frequently misstated point about that oxidation is worth stating correctly: the reduced form is not auto-oxidised by molecular oxygen in the absence of a catalyst. The reaction that actually consumes it in ordinary buffers is catalysed by trace copper, proceeding through a copper-thiol complex, and superoxide will oxidise it to the disulfide along with a sulfonate by-product. This is why buffers for thiol work are chelated and why glassware and water quality matter more here than for most compounds in the catalogue.

It is used on the bench as a redox reference in reduced-to-oxidised ratio assays, as the substrate for peroxidase and transferase enzyme assays, and as a reducing additive in buffers. Supplier technical data give aqueous solubility around 20 mg/mL and advise against holding aqueous solutions beyond a day; a much higher solubility figure that appears in one public database carries no supporting reference and should not be relied on.