Glutathione is a tripeptide, γ-L-glutamyl-L-cysteinyl-glycine, with the formula C10H17N3O6S and a molecular weight of 307.33 g/mol (PubChem CID 124886, CAS 70-18-8). It is unusual among peptides because glutamate is joined to cysteine through its side-chain γ-carboxyl rather than the usual α-carboxyl, and its chemistry is dominated by the free thiol of the cysteine. In mammalian cells it is the main non-protein thiol, present at millimolar concentrations, and it interconverts between a reduced form (GSH) and a disulfide-linked oxidized form (GSSG).
That combination of a small size, a protected backbone and a reactive thiol makes glutathione one of the most studied molecules in redox biochemistry. This profile covers its identity data, history, structure, how cells make and use it at the molecular level, how it is measured, and how to handle it as a laboratory reagent.
Identity
| Property | Reduced glutathione (GSH) | Glutathione disulfide (GSSG) |
|---|---|---|
| Sequence | γ-Glu-Cys-Gly | Two γ-Glu-Cys-Gly chains linked by a Cys-Cys disulfide |
| Length | 3 residues | 2 × 3 residues |
| Molecular formula | C10H17N3O6S | C20H32N6O12S2 |
| Molecular weight | 307.33 g/mol | 612.6 g/mol |
| Monoisotopic mass | 307.0838 Da | 612.1520 Da |
| CAS number | 70-18-8 | 27025-41-8 |
| PubChem CID | 124886 | 65359 |
| Computed XLogP | −4.5 | −9.1 |
| Functional group | Free thiol (−SH) on Cys | Disulfide (−S−S−) |
The IUPAC name PubChem gives for GSH is (2S)-2-amino-5-[[(2R)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2-yl]amino]-5-oxopentanoic acid. The (2R) label on cysteine is not a D-amino acid; it is L-cysteine, whose sulfur changes the priority rules that assign R and S. Both computed XLogP values are strongly negative, consistent with a highly polar, water-soluble molecule, and PubChem lists an aqueous solubility of about 292.5 mg/mL for GSH.
Glutathione is listed in the Anhydrolabs catalog as glutathione.
History
The molecule was noticed long before its structure was known. In 1888 de Rey-Pailhade described a sulfur-reactive substance in blood, sheep brain, fish and vegetables and called it "philothion", before the peptide bond had even been formally proposed. Thirty-three years later, in 1921, Frederick Gowland Hopkins reported it in the Biochemical Journal as an autoxidizable constituent of the cell, and the substance was renamed glutathione. Alton Meister later contributed much of the structural and enzymological work on its synthesis and breakdown and wrote a detailed history of the field. Those early descriptions already captured the two properties that still define it: it reacts readily with oxygen, and it is found in almost every cell.
Structure: why the γ-bond matters
A normal peptide bond joins the α-carboxyl of one amino acid to the α-amino group of the next. In glutathione, the first bond is formed from glutamate's side-chain carboxyl, the γ-carboxyl, instead. The second bond, between cysteine and glycine, is an ordinary α-peptide bond. If you are new to peptide nomenclature, what peptides are explains the standard backbone this departs from.
That one change has two consequences:
- Resistance to ordinary peptidases. Most peptidases recognize α-peptide bonds. The γ-glutamyl bond is not a substrate for them, which protects glutathione from routine intracellular breakdown. It is cleaved instead by a dedicated membrane enzyme, γ-glutamyl transpeptidase, which releases the cysteinyl-glycine dipeptide for ordinary peptidases to finish.
- Non-ribosomal origin. Because the γ-bond cannot be made by the ribosome, glutathione is not encoded by any gene. It is assembled by two enzymes, described below.
The reactive part of the molecule is the cysteine thiol. It can donate an electron and pair with a second glutathione to form GSSG, attack electrophiles to form conjugates, and form mixed disulfides with cysteine residues on proteins. Glutathione is small enough that it is usually classed as a peptide rather than anything larger; the post on peptides vs proteins sets out where that line is drawn.
How cells make and recycle glutathione
Glutathione synthesis takes two ATP-dependent steps, both described in detail in the 2023 review by Georgiou-Siafis and Tsiftsoglou:
| Step | Enzyme | Reaction | Notes |
|---|---|---|---|
| 1 | Glutamate-cysteine ligase (GCL) | Glu + Cys → γ-Glu-Cys | Rate-limiting; a heterodimer of catalytic and regulatory subunits; competitively inhibited by GSH |
| 2 | Glutathione synthetase (GSS) | γ-Glu-Cys + Gly → γ-Glu-Cys-Gly | Ordinary α-peptide bond to glycine |
| Recycling | Glutathione reductase | GSSG + NADPH → 2 GSH | Cytosolic, mitochondrial and nuclear forms |
| Breakdown | γ-Glutamyl transpeptidase | Cleaves the γ-bond | Membrane-bound; releases Cys-Gly |
Both synthetic enzymes have a low Km for ATP, so synthesis can continue under energy-limited conditions. Of the three amino acids, cysteine is present at the lowest concentration and is the rate-limiting substrate. In cell and animal research, buthionine sulfoximine (BSO), an inhibitor of GCL, is the standard tool for depleting glutathione experimentally, which lets investigators compare cells with and without a normal glutathione pool.
Concentrations and the GSH/GSSG ratio
Glutathione is present inside cells at concentrations far higher than most metabolites. The figures below are from the same review and describe mammalian systems.
| Location | Reported glutathione level |
|---|---|
| Cells, general | 1 to 5 mM |
| Liver | Up to about 10 mM |
| Red blood cells | 0.4 to 3.0 mM |
| Plasma | About 3.6 µM |
| Share of non-protein thiols (mammals) | More than 90% |
| Typical GSH:GSSG ratio | Around 100:1 |
The ratio matters as much as the total. Because GSSG is continually reduced back to GSH by glutathione reductase at the expense of NADPH, the GSH/GSSG pair acts as a redox buffer, with a calculated redox potential between about −260 and −150 mV in different tissues. A shift toward GSSG is one of the most widely used readouts of oxidative stress in cell models.
Molecular roles studied in the literature
Research on glutathione tends to fall into four areas, each defined by what the thiol does:
- Peroxide reduction. Glutathione is the cofactor for the glutathione peroxidases, a family of eight isoenzymes that reduce hydrogen peroxide and lipid peroxides to water or alcohols, oxidizing two GSH to one GSSG in the process.
- Conjugation. Glutathione S-transferases attach GSH through its sulfur to electrophilic compounds, forming thioether conjugates. Substrates studied include xenobiotics such as NAPQI (the reactive metabolite of acetaminophen) and endogenous compounds such as leukotrienes and prostaglandins.
- Protein S-glutathionylation. Glutathione forms mixed disulfides with reactive cysteine residues on proteins. This reversible modification protects those residues from irreversible oxidation and changes the activity of some proteins, and it is a research field of its own.
- Redox signaling. Together with the NADPH/NADP+ and thioredoxin systems, the GSH/GSSG couple sets the reducing environment of the cytosol and organelles, which influences the thiol-sensing proteins that regulate gene expression. The nicotinamide side of that arithmetic is why NAD+ is a common companion reagent in redox experiments: NADPH is regenerated from NADP+, and both pools trace back to nicotinamide metabolism.
Taken together, these are what the word antioxidant means at the molecular level, and the role of glutathione in it is mostly indirect. Glutathione peroxidases use it to scavenge reactive oxygen species and reactive nitrogen species, and the transferases use it to conjugate electrophiles, the biochemical basis of detoxification in hepatic and other cells. Intracellular glutathione does not simply neutralize free radicals one for one. It is an endogenous redox buffer that enzymes draw on and NADPH restores, which is how cells keep redox homeostasis under oxidative stress.
Most of this work uses cultured cells, isolated enzymes and animal models, with glutathione measured rather than added. When purified glutathione is used as a reagent, it is typically as an assay standard, an enzyme substrate for glutathione S-transferase or peroxidase assays, a redox buffer component, or a reference compound for analytical methods.
Measuring glutathione
Because GSH oxidizes easily during sample handling, measurement methods are designed around keeping the two forms apart:
- Enzymatic recycling. Tietze's 1969 enzymatic method quantifies total and oxidized glutathione at nanogram levels, and variations of it are still widely used.
- HPLC and LC-MS. Chromatographic methods separate GSH, GSSG and conjugates; mass spectrometry confirms identity at 307.08 Da for GSH and 612.15 Da for GSSG (monoisotopic).
- Thiol blocking. Samples are often treated with a thiol-alkylating reagent immediately after collection so GSH cannot oxidize to GSSG before analysis.
For reagent-grade material, the certificate of analysis reports identity by mass spectrometry and purity by HPLC, where GSSG is the most likely related substance.
Handling glutathione in the laboratory
Glutathione's free thiol sets the rules for working with it. The general guidance in how to reconstitute peptides applies, with a few points specific to a thiol:
| Consideration | Practice |
|---|---|
| Dry storage | Frozen at −4 °F (−20 °C), protected from light, per the storage and handling page |
| Opening | Let the sealed vial reach room temperature first so moisture does not condense on the powder |
| Solvent | Water or aqueous buffer; highly soluble, so no organic co-solvent is needed |
| Oxygen | Use degassed solvent and keep headspace small; the thiol autoxidizes to GSSG |
| pH | Slightly acidic stocks oxidize more slowly than neutral or basic ones |
| Metals | Trace transition metals catalyze thiol oxidation; use clean glassware and high-purity water |
| DMSO | Avoid; it oxidizes thiols and is used deliberately to form disulfides |
| Reconstituted storage | Refrigerated at 39 °F (4 °C), aliquoted; about four weeks, per the storage page |
| Checking a stored stock | HPLC or an enzymatic assay for the GSH:GSSG ratio |
The main change in a stored glutathione solution is oxidation to GSSG rather than breakdown of the backbone, since the γ-bond is chemically robust. That makes a stock easy to check: the fraction present as GSSG tells you how far it has oxidized. The general principles for how long peptide solutions keep, and why, are in how long do peptides last.
Reagent grade versus dietary supplement grade
Glutathione is also sold in bulk as a dietary supplement ingredient, and the grades are not interchangeable. In a June 2019 compounding alert, FDA reported that L-glutathione powder labeled "Caution: Dietary Supplement" had been used to make sterile preparations, and that every sample an FDA laboratory tested contained excessive bacterial endotoxin, in some cases five times the appropriate limit. For glutathione research in cell culture, that is a reason to use a lot supplied for research use with its own COA, and to read what the COA does and does not test.
Anhydrolabs supplies glutathione as a lyophilized powder in vacuum-sealed vials, and as 10-vial kits for groups that use it across many assays. It is sold for laboratory research only.
