Glutathione is a tripeptide of glutamate, cysteine and glycine and the most abundant low molecular weight thiol in most cells, present at one to ten millimolar in cytosol. It is unusual among peptides in that its first bond is formed through the gamma carboxyl of glutamate instead of the alpha, which makes it invisible to ordinary aminopeptidases. Almost all of its chemistry runs through the sulfhydryl on the central cysteine.
Key facts
| Type | Gamma-glutamyl tripeptide, the principal cellular thiol antioxidant |
| Sequence | gamma-L-glutamyl-L-cysteinyl-glycine |
| Molecular formula | C10H17N3O6S |
| Molecular weight | 307.32 g/mol for the reduced form |
| CAS number | 70-18-8 for reduced glutathione |
| Oxidised form | GSSG, a disulfide-linked dimer of 612.6 g/mol |
| Typical cytosolic concentration | 1 to 10 mM, with a GSH to GSSG ratio above 100 to 1 |
| Synonyms | GSH, L-glutathione reduced, gamma-Glu-Cys-Gly |
Structure and chemistry
The gamma linkage is the defining structural feature. Glutamate joins cysteine through its side chain carboxyl and not through the alpha carboxyl that forms a standard peptide bond, so the tripeptide cannot be cleaved by aminopeptidases and is instead degraded by gamma-glutamyl transpeptidase at the cell surface. That single bond is why glutathione survives in extracellular fluid long enough to serve as a cysteine transport form, and why its turnover runs through a dedicated enzyme cycle.
Synthesis is enzymatic and takes two ATP-dependent steps. Glutamate-cysteine ligase joins glutamate to cysteine, a step inhibited by the product and rate limiting in most cells, and glutathione synthetase adds glycine. Cysteine availability sets the ceiling on the first step, which is why cysteine precursors are the usual experimental lever on cellular glutathione content.
The reduced and oxidised forms differ by two hydrogens across a disulfide. Two GSH molecules oxidise to one GSSG of 612.6 g/mol. Reduced glutathione in solution oxidises spontaneously in air, faster at neutral to alkaline pH and faster again in the presence of trace copper or iron, so a solution left on the bench is a mixture within hours. Analytical methods that do not derivatise the free thiol immediately, usually with N-ethylmaleimide or monobromobimane, measure a ratio that has shifted during sample handling.
Mechanism of action
The functional unit is the GSH to GSSG couple, which sets the reduction potential of the cytosol at roughly minus 240 millivolts. Glutathione peroxidases use two GSH to reduce hydrogen peroxide and organic hydroperoxides to water and alcohols, producing GSSG, and glutathione reductase regenerates GSH using NADPH from the pentose phosphate pathway. The ratio of the two forms, and not the absolute concentration, is what reports redox state in a cell.
Beyond peroxide handling, glutathione S-transferases conjugate GSH to electrophiles for export through the mercapturic acid pathway, glutaredoxins use it to reverse protein S-glutathionylation, and the tripeptide chelates and buffers reactive metals. S-glutathionylation of cysteine residues on signalling proteins is now treated as a reversible post-translational modification in its own right, so glutathione sits in the middle of redox signalling and not only in the disposal of oxidants.
Research applications
- GSH to GSSG ratio quantification by enzymatic recycling assay or LC-MS with immediate thiol derivatisation.
- Oxidative stress models in cell culture using buthionine sulfoximine to deplete glutathione by inhibiting glutamate-cysteine ligase.
- Glutathione S-transferase activity and conjugate formation assays with model electrophile substrates.
- Protein S-glutathionylation mapping by redox proteomics with thiol blocking and selective reduction.
- Redox potential measurement in live cells using genetically encoded roGFP and Grx1-roGFP2 sensors.
- Metal chelation and Fenton chemistry studies examining copper and iron buffering by the free thiol.
Compounds studied around cellular maintenance and redox balance sit in NuVion’s Cellular Ageing category.
Handling in the laboratory
Reduced glutathione is supplied as a lyophilised powder or crystalline solid and dissolves readily in water, giving an acidic solution that is more stable than a neutral one. Reconstitution is with bacteriostatic or sterile water added slowly and swirled. The reconstitution calculator converts vial content and diluent volume into a stock concentration. Stock intended for redox work is prepared fresh on the day, since the assumption that a stored aliquot is fully reduced is rarely correct.
Trace metals accelerate air oxidation, so buffers for this compound are treated with a chelator such as EDTA or diethylenetriaminepentaacetic acid, and degassed water is used where the work allows it. Solutions are kept acidic and cold until use, headspace in the vial is minimised, and any sample intended for a GSH to GSSG measurement has its free thiol blocked at the moment of collection. Most inconsistent glutathione data traces back to oxidation between sampling and analysis and not to the biology under study.
Testing and supply from NuVion
NuVion supplies a tested range across cellular maintenance and redox biology, including NAD+, SS-31, MOTS-C and Epithalon. That range is manufactured at a GMP-audited facility and independently tested by Janoshik Analytical, with purity determined by RP-HPLC and identity confirmed by mass spectrometry, and the certificates are published in the Certificate of Analysis library. Glutathione is not part of the current catalogue and this page is a reference profile.
Related compounds
Within cellular energetics and redox handling, NAD+ covers the pyridine nucleotide pool that supplies reducing equivalents through NADPH, and SS-31 covers the cardiolipin-binding mitochondrial peptide studied on electron transport efficiency. MOTS-C covers the mitochondrial derived peptide arm and Epithalon the telomere and pineal work. Background on peptide bond geometry, including why the gamma linkage behaves differently, is in the peptide bond.
Frequently asked questions
Why is glutathione called a gamma-glutamyl peptide?
Because glutamate is joined to cysteine through its side chain carboxyl and not through the alpha carboxyl used in ordinary peptide bonds. That geometry makes the bond unrecognisable to aminopeptidases, so the tripeptide is stable outside the cell and is broken down only by gamma-glutamyl transpeptidase at the membrane.
What is the difference between GSH and GSSG?
GSH is the reduced tripeptide with a free sulfhydryl, molecular weight 307.32. GSSG is two of those molecules joined through a disulfide, molecular weight 612.6. Healthy cytosol holds a ratio above one hundred to one in favour of the reduced form, and the shift in that ratio is the standard reporter of oxidative stress.
Why does a glutathione solution lose activity on the bench?
The free thiol oxidises in air, and the reaction is faster at neutral or alkaline pH and faster again with trace copper or iron present. A solution prepared and left uncovered is a GSH and GSSG mixture within hours. Fresh preparation, acidic pH, a metal chelator in the buffer and minimal headspace all slow it.
Is glutathione listed in the Australian Register of Therapeutic Goods?
Reduced glutathione supplied as a laboratory chemical for in vitro research is not an ARTG-listed product and has not been assessed by the Therapeutic Goods Administration for quality, safety or efficacy. Material of this class is supplied in Australia for laboratory use only.
Research use only. The compound described on this page is discussed as a laboratory chemical used in in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the Therapeutic Goods Administration for quality, safety or efficacy. It is not for human or veterinary use, and nothing on this page is a representation about therapeutic use.
