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Glutathione: A Research Overview of the Gamma-Glutamyl Tripeptide

By The Bionova Club Team

Research use only. Bionova products are sold strictly for in-vitro and laboratory research by qualified professionals. They are not drugs, food, or cosmetics, are not approved by the FDA, and are not for human or veterinary use. Nothing in this article is medical advice or a claim about effects in people.

Disclosure: Bionova Club sells glutathione in a 1500 mg vial, linked below.

Glutathione is the most abundant non-protein thiol in mammalian tissues (Lu, 2013). It is a tripeptide, three amino acids long, but it is an unusual one: one of its two linking bonds is not a standard peptide bond. That detail explains a lot about how it behaves in the lab.

This overview covers what glutathione is chemically, how it was discovered, what its reduced and oxidized forms are, how laboratories measure it, and how to keep the sealed dry material in good condition.

What is glutathione?

Glutathione (often abbreviated GSH for the reduced form) is the tripeptide γ-L-glutamyl-L-cysteinylglycine. It is built from glutamic acid, cysteine and glycine (PubChem CID 124886).

  • Sequence: γ-Glu-Cys-Gly (written H-γGlu-Cys-Gly-OH)
  • Molecule type: Tripeptide with one gamma-glutamyl linkage
  • Molecular formula: C₁₀H₁₇N₃O₆S
  • Molecular weight: ~307.3 g/mol
  • CAS number: 70-18-8
  • Key functional group: Free thiol (–SH) on the cysteine residue
  • Oxidized form: Glutathione disulfide (GSSG), C₂₀H₃₂N₆O₁₂S₂, ~612.6 g/mol
  • Bionova format: Glutathione 1500 mg, supplied lyophilized

Formula and weight are from PubChem (GSH, CID 124886; GSSG, CID 65359). The formula, CAS number and sequence match Bionova's product page.

The gamma-glutamyl bond

In an ordinary peptide, each amino acid is joined through the carboxyl group on its central (alpha) carbon. In glutathione, glutamic acid is joined to cysteine through the carboxyl group at the end of its side chain, the gamma carboxyl, instead. The cysteine–glycine bond is a normal peptide bond.

This gamma linkage matters because most peptidases do not recognize it. Glutathione is broken down by a dedicated enzyme, γ-glutamyl transpeptidase, rather than by general peptide-cutting enzymes. The classic review of glutathione biochemistry by Meister and Anderson describes the gamma-glutamyl cycle in detail (Meister & Anderson, 1983, Annual Review of Biochemistry).

For a refresher on standard peptide bonds and how sequences are written, see What are peptides? A primer for researchers.

A short history

In 1921 Frederick Gowland Hopkins reported a sulfur-containing substance in yeast and animal tissues that was readily oxidized and reduced, and he named it glutathione (Hopkins, 1921, Biochemical Journal). Hopkins first described it as a dipeptide of glutamic acid and cysteine, and he published a reinvestigation of the substance in 1929 (Hopkins, 1929, Journal of Biological Chemistry).

Its tripeptide structure, with glycine as the third residue, was confirmed by chemical synthesis in 1935, when Charles Harington and Thomas Mead reported a synthesis of glutathione (Harington & Mead, 1935, Biochemical Journal).

Reduced vs oxidized glutathione (GSH vs GSSG)

The chemistry of glutathione centers on the thiol group of its cysteine residue.

  • GSH (reduced) carries a free –SH group.
  • GSSG (oxidized) forms when two GSH molecules are joined through a disulfide (–S–S–) bond. It is roughly twice the mass of GSH.

The two forms interconvert. In biochemical systems the enzyme glutathione reductase converts GSSG back to GSH, and the ratio of the two is widely used as a laboratory readout of redox state (Forman et al., 2009, Molecular Aspects of Medicine).

What has been studied (biochemistry only)

Glutathione's literature is enormous. In biochemical and cell-based research it is described in three main ways:

  1. The most abundant non-protein thiol in mammalian cells. A review of its synthesis describes GSH as present in all mammalian tissues and made in the cytosol in two enzyme steps, by glutamate-cysteine ligase and then glutathione synthetase (Lu, 2013, Biochimica et Biophysica Acta).
  2. A cofactor and substrate for enzymes. It is used by enzyme families such as the glutathione peroxidases, which reduce peroxides, and the glutathione S-transferases, which attach glutathione to electrophilic compounds (Forman et al., 2009).
  3. A redox marker. Because GSH and GSSG interconvert, their levels and ratio are measured in cell and tissue samples to characterize oxidative conditions in an experiment (Forman et al., 2009).

These are descriptions of biochemistry in research systems. They are not claims about what glutathione does when taken by people.

How laboratories measure glutathione

Several standard analytical methods exist, and they are worth knowing for anyone checking the identity or condition of a sample.

  • Ellman's reagent (DTNB). Introduced in 1959, 5,5′-dithiobis(2-nitrobenzoic acid) reacts with free thiol groups to give a yellow product that can be read on a spectrophotometer (Ellman, 1959, Archives of Biochemistry and Biophysics).
  • Enzymatic recycling assay. Tietze's 1969 method couples DTNB with glutathione reductase so that total glutathione (GSH plus GSSG) can be measured at very low amounts (Tietze, 1969, Analytical Biochemistry). A widely used step-by-step protocol for measuring GSH and GSSG separately was published in Nature Protocols in 2006 (Rahman et al., 2006).
  • Chromatography and mass spectrometry. As with other research peptides, HPLC is used to assess purity and mass spectrometry to confirm identity. For glutathione, a mass near 307 Da points to GSH and a mass near 612 Da points to GSSG.

One practical point from the measurement literature: GSH can oxidize to GSSG during sample handling, which is why published methods pay close attention to how samples are prepared (Forman et al., 2009).

Glutathione vs NAD+

Glutathione and NAD+ are often searched together because both appear in redox biochemistry. They are different kinds of molecule:

  • Molecule type
    • Glutathione: Tripeptide
    • NAD+: Coenzyme (dinucleotide)
  • Contains amino acids?
    • Glutathione: Yes: glutamic acid, cysteine, glycine
    • NAD+: No
  • Redox chemistry
    • Glutathione: Thiol/disulfide (GSH ⇄ GSSG)
    • NAD+: Hydride transfer (NAD+ ⇄ NADH)
  • Molecular weight
    • Glutathione: ~307.3 g/mol
    • NAD+: ~663.4 g/mol

Our article on NAD+ in the lab covers NAD+ chemistry and dry storage.

Glutathione vs GHK-Cu

Both are tripeptides, but they share no residues and behave very differently. GHK (Gly-His-Lys) is known for binding copper, while glutathione's chemistry is driven by its cysteine thiol. See our GHK-Cu research overview.

Storage notes for lyophilized glutathione

The thiol group that defines glutathione is also its main weak point: it oxidizes in the presence of oxygen and moisture.

  • Keep it sealed, cold and dry. Store the lyophilized powder in its original sealed vial, away from light and moisture.
  • Warm before opening. Let a sealed vial reach room temperature before opening so moisture does not condense on the powder.
  • Check the appearance. Material that looks wet, clumped or discolored should be reported to the supplier.
  • Follow the label. Store as directed on the product label and batch documentation.

This guide covers only the sealed, dry material. It does not cover preparing solutions.

Key takeaways

  • Glutathione is the tripeptide γ-Glu-Cys-Gly (C₁₀H₁₇N₃O₆S, ~307.3 g/mol).
  • Its glutamate is linked through the side-chain (gamma) carboxyl, so most peptidases do not cut it.
  • The cysteine thiol drives its chemistry: two GSH molecules form the disulfide GSSG.
  • Hopkins named it in 1921, and its structure was confirmed by synthesis in 1935.
  • Laboratories measure it with Ellman's reagent, enzymatic recycling assays, HPLC and mass spectrometry.
  • Bionova sells glutathione in a 1500 mg vial. It is a research material only and is not approved for human use.

Frequently asked questions

Is glutathione a peptide?

Yes. It is a tripeptide of glutamic acid, cysteine and glycine. It is unusual because the glutamate is joined through its side-chain carboxyl group instead of the usual alpha carboxyl.

What is the difference between GSH and GSSG?

GSH is reduced glutathione, with a free thiol group. GSSG is oxidized glutathione, two GSH molecules joined by a disulfide bond.

Who discovered glutathione?

Frederick Gowland Hopkins named it in 1921. Its tripeptide structure was established later in the 1920s and confirmed by chemical synthesis in 1935.

How is glutathione measured in the lab?

Common methods include Ellman's reagent (DTNB), the Tietze enzymatic recycling assay, HPLC and mass spectrometry.

What is the difference between glutathione and NAD+?

Glutathione is a tripeptide whose chemistry depends on a thiol group. NAD+ is a coenzyme with no amino acids that transfers hydride in redox reactions.

How should lyophilized glutathione be stored?

Keep the sealed vial cold, dry and away from light, let it reach room temperature before opening, and store it as directed on the product label.

Shop for your research: Glutathione for laboratory research. Use code CLUB10 at checkout for 10% off.

References

  1. Hopkins FG. On an Autoxidisable Constituent of the Cell. Biochem J. 1921. https://pubmed.ncbi.nlm.nih.gov/16742989/
  2. Harington CR, Mead TH. Synthesis of glutathione. Biochem J. 1935. https://pubmed.ncbi.nlm.nih.gov/16745829/
  3. Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983. https://pubmed.ncbi.nlm.nih.gov/6137189/
  4. Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles, measurement, and biosynthesis. Mol Aspects Med. 2009. https://pubmed.ncbi.nlm.nih.gov/18796312/
  5. Lu SC. Glutathione synthesis. Biochim Biophys Acta. 2013. https://pubmed.ncbi.nlm.nih.gov/22995213/
  6. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959. https://pubmed.ncbi.nlm.nih.gov/13650640/
  7. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969. https://pubmed.ncbi.nlm.nih.gov/4388022/
  8. Rahman I, Kode A, Biswas SK. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc. 2006. https://pubmed.ncbi.nlm.nih.gov/17406579/
  9. PubChem. Glutathione, CID 124886. https://pubchem.ncbi.nlm.nih.gov/compound/124886
  10. PubChem. Glutathione Disulfide, CID 65359. https://pubchem.ncbi.nlm.nih.gov/compound/65359
  11. Hopkins FG. On glutathione: a reinvestigation. J Biol Chem. 1929. https://doi.org/10.1016/S0021-9258(18)77062-2

For laboratory research use only. Not for human consumption.