Masses calculated from residue masses; the γ-linkage does not change mass or formula.
An unusual tripeptide
Glutathione is γ-L-glutamyl-L-cysteinyl-glycine, a tripeptide present in almost every animal and plant cell and in many bacteria, usually at millimolar concentrations. That makes it by far the most abundant low-molecular-weight thiol in the cell.
Its structure has one feature that sets it apart from an ordinary peptide. The glutamate is linked to cysteine not through its α-carboxyl group but through the carboxyl at the end of its side chain, the γ-carboxyl. This is an isopeptide bond, described in the guide to the peptide bond. Most peptidases are built to recognise α-linked bonds and cannot cleave it, which protects glutathione from the ordinary turnover of intracellular peptides. Only a dedicated enzyme, γ-glutamyl transpeptidase on the outer surface of cells, can break it.
Because the γ-bond has the same atoms as an α-bond, the mass is unaffected. Entering ECG in the MW calculator gives the correct mass and formula for glutathione, even though the calculator assumes an α-linkage. Its ionisable groups are also the same set: two carboxyls, one amine and the thiol. At pH 7.4 glutathione carries a net charge of about −1, from its two carboxylates and one ammonium group. Published values for its thiol pKa are around 8.7–9.0, somewhat higher than the generic cysteine value used by the calculator, so the calculator slightly overestimates the thiolate fraction.
Synthesis and turnover
Glutathione is not made on the ribosome. Two ATP-dependent enzymes assemble it in the cytoplasm:
- Glutamate–cysteine ligase joins the γ-carboxyl of glutamate to cysteine. This is the rate-limiting step, and the enzyme is inhibited by glutathione itself, a simple feedback loop that keeps concentrations stable.
- Glutathione synthetase adds glycine.
The availability of cysteine usually limits how fast glutathione can be made. Glutathione is exported from cells, broken down outside them by γ-glutamyl transpeptidase and dipeptidases, and its amino acids are taken back up and reused. The liver is the main producer and exports large amounts into the blood and bile.
The redox couple GSH/GSSG
Glutathione exists in a reduced form, GSH, with a free thiol, and an oxidised form, glutathione disulfide or GSSG, in which two molecules are joined by a disulfide bond. The reaction transfers two electrons:
In the cytoplasm of a healthy cell, the enzyme glutathione reductase, using electrons from NADPH, keeps the couple strongly reduced: GSH usually outnumbers GSSG by 100 to 1 or more. The endoplasmic reticulum, where secreted proteins form their disulfide bonds, is kept far more oxidising.
The reducing power of the couple is expressed as a redox potential. Its standard value at pH 7 and 25 °C is about −240 mV. Because two molecules of GSH are consumed for each GSSG formed, the actual potential depends on the square of the GSH concentration:
The first two lines show something that surprises many students. At the same ratio of GSH to GSSG, diluting the total pool makes the potential less reducing. Unlike simpler redox couples, the glutathione potential is not determined by the ratio alone, which is why cells regulate the total amount of glutathione as well as its oxidation state.
What glutathione does
Removing peroxides
Glutathione peroxidases, many of which contain the unusual amino acid selenocysteine in their active site, use two molecules of GSH to reduce hydrogen peroxide to water and lipid hydroperoxides to alcohols, producing GSSG. Glutathione reductase then regenerates GSH with NADPH. This cycle is one of the main defences of cells against oxidative damage, and it links glutathione to the pentose phosphate pathway, the main source of NADPH.
Detoxification
Glutathione S-transferases attach glutathione through its sulfur atom to a wide range of reactive, electrophilic compounds, including many drug metabolites and environmental chemicals. The conjugates are more water-soluble and are exported from the cell, then processed further, mainly in the kidney, into mercapturic acids that are excreted in urine.
A well-known illustration is paracetamol (acetaminophen). A small fraction of every dose is converted in the liver into a reactive metabolite, NAPQI, that is normally neutralised by glutathione. When very large amounts are ingested, the glutathione pool is exhausted and the metabolite damages liver cells. The standard antidote, N-acetylcysteine, works mainly by supplying cysteine for glutathione synthesis.
Maintaining protein thiols
Glutathione keeps cysteine residues of cytoplasmic proteins in their reduced form. It can also form mixed disulfides with protein cysteines, a reversible modification called S-glutathionylation, which protects those cysteines from irreversible oxidation during oxidative stress and can regulate enzyme activity.
Glutathione in the laboratory
- Redox buffers for oxidative folding. Mixtures of GSH and GSSG, typically in a ratio of around 10:1, allow disulfide-rich peptides to form and reshuffle their disulfides until the native pattern is reached. The method is described in the guide to disulfide bonds.
- GST fusion proteins. Glutathione S-transferase from the parasite Schistosoma japonicum is widely used as a tag: proteins fused to it bind to glutathione immobilised on agarose beads and are eluted with free glutathione.
- Measurement. Total glutathione is commonly measured with an enzymatic recycling assay using glutathione reductase and Ellman's reagent. GSSG is measured after free GSH has been blocked, and the ratio is sensitive to oxidation during sample preparation, which must be prevented.
A note on glutathione supplements
Glutathione is sold widely as an oral supplement. Because it is a peptide, most of an oral dose is broken down in the gut and the blood by γ-glutamyl transpeptidase and dipeptidases, and cells cannot take up intact glutathione efficiently in any case; they make their own from its component amino acids. Studies of whether oral glutathione raises levels in blood or tissues have produced mixed results. The biochemistry described on this page does not in itself establish any health benefit from supplementation, and questions about personal use are best discussed with a qualified professional.
Frequently asked questions
Why is glutathione linked through the γ-carboxyl?
The γ-linkage makes it resistant to ordinary peptidases, allowing it to accumulate to millimolar levels without being degraded. It also leaves the α-amino and α-carboxyl groups of the glutamate free, and both contribute to recognition by the enzymes that use glutathione.
Is GSSG harmful?
Not in itself, but a rising proportion of GSSG signals oxidative stress. Cells export GSSG or reduce it quickly, and the GSH:GSSG ratio is widely used as an indicator of the cellular redox state.
Do all organisms use glutathione?
Most eukaryotes and many bacteria do, but not all. Some bacteria use other thiols instead, such as mycothiol in mycobacteria and bacillithiol in some Gram-positive bacteria, and trypanosomes use trypanothione, a conjugate of two glutathione molecules with spermidine.
What is the molecular weight of glutathione?
Reduced glutathione (GSH), C₁₀H₁₇N₃O₆S, has an average molecular weight of 307.33 and a monoisotopic mass of 307.0838 Da. The disulfide GSSG is 612.63 average and 612.1519 Da monoisotopic, two GSH molecules minus two hydrogen atoms.
References
- Meister A, Anderson ME (1983) Glutathione. Annual Review of Biochemistry 52:711–760.
- Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radical Biology & Medicine 30:1191–1212.
- Forman HJ, Zhang H, Rinna A (2009) Glutathione: overview of its protective roles, measurement, and biosynthesis. Molecular Aspects of Medicine 30:1–12.
- Hwang C, Sinskey AJ, Lodish HF (1992) Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257:1496–1502.