Glutathione

Glutathione is a tripeptide, but not an ordinary one. Two features set it apart from every other peptide in this library: the glutamate is joined through its side-chain carboxyl rather than its α-carboxyl, and the molecule’s entire chemistry is dominated by a single free thiol.

The gamma linkage

In an ordinary peptide bond, the α-carboxyl of one residue joins the α-amino of the next. In glutathione, the glutamate contributes its γ-carboxyl — the one on the side chain — to the bond with cysteine. The Cys–Gly bond is a normal α-linkage.

This is not a notational curiosity. Peptidases recognise α-linkages; the γ-bond is not a substrate for ordinary peptidases, which is why glutathione is not degraded by the proteolytic machinery that would rapidly clear a conventional tripeptide. Writing the compound as “Glu-Cys-Gly” without the γ loses the single most important structural fact about it.

Identity and classification

Glutathione is classified by biochemical role — a thiol tripeptide and enzyme cofactor — rather than by receptor target. It is not a receptor ligand, and no target is claimed. It is also used as an elution reagent in glutathione-S-transferase fusion protein purification and as an analytical standard.

GSH and GSSG — and an arithmetic trap

The oxidised form, glutathione disulfide (GSSG), is two glutathione molecules joined by a disulfide bridge between their cysteine thiols.

GSSG is 612.6 g/mol, not 614.6. Forming the S–S bond removes one hydrogen from each thiol, so the disulfide is two daltons lighter than twice the monomer. Doubling 307.32 gives 614.64, which is wrong, and it appears on listings often enough to be a useful diagnostic of whether anyone checked.

Because GSH and GSSG are different compounds with different registry numbers, a specification must state which form it describes. “Glutathione” without qualification is ambiguous; this material is the reduced form.

Specification and characterisation

• Free thiol content by Ellman’s assay. DTNB reacts with free thiols to give a chromophore measured at 412 nm. This is the most informative single number for reduced glutathione, because it measures the functional group directly. A material that is 99% pure by HPLC but substantially oxidised will show low free thiol — and it is the thiol, not the mass, that the reagent exists to provide.
• GSSG content. The oxidised dimer is the primary impurity and is chromatographically and mass-resolvable from the monomer.
• HPLC purity and assay against a reference standard.
• Optical rotation. Glutathione is a defined stereoisomer and specific rotation is a classical identity check that also reports on stereochemical integrity.
• Water content by Karl Fischer, and residue on ignition for inorganic residue.
• Heavy metals. Relevant for a thiol, which chelates metals readily — and trace transition metals catalyse the very oxidation the material is prone to.

Handling and stability

Store the sealed solid dry. Reference sources differ on temperature: Sigma specifies 2–8 °C; Cayman specifies −20 °C with a stated stability of at least four years. Either is defensible for the dry solid; the disagreement is worth knowing about when comparing certificates.

Solutions are the problem, not the solid. Free cysteine thiols oxidise readily above pH 7, and reference catalogues do not recommend storing aqueous glutathione solutions beyond one day. Where solution work is required, use freshly prepared, degassed, acidic-to-neutral buffers; trace metal contamination markedly accelerates oxidation, so chelator-treated buffers are a reasonable precaution.

The lyophilised solid is hygroscopic. Equilibrate the vial to room temperature in a desiccator before opening and reseal promptly.

Related compounds in this library

AOD-9604 is the other sulfur-redox compound here — a peptide with a formed intramolecular disulfide, where glutathione is the free thiol. GHK-Cu is the other tripeptide, and the two make an instructive pair: both are small tripeptides whose behaviour is governed almost entirely by one functional feature, a metal-binding site in one case and a redox-active thiol in the other.

References

Chemical, specification and analytical sources. This list is deliberately limited to chemistry and analytical references.

• Sigma-Aldrich G4251, L-glutathione reduced — specification, appearance, solubility, storage
• Cayman Chemical 10007461, L-glutathione reduced — formula, purity, storage, solution stability
• Cayman Chemical 35825, glutathione disulfide — GSSG formula, mass, registry
• PubChem CID 65359, glutathione disulfide — independent mass confirmation
• Bachem, Handling and Storage Guidelines for Peptides — cysteine oxidation above pH 7, degassed buffers, hygroscopicity

Cosmic Aminos supplies this material as a laboratory reagent for in-vitro research use only. It is not a drug, food, cosmetic or dietary supplement, is not approved by the Food and Drug Administration for any use, and is not for human or veterinary consumption. No dosing, administration or preparation guidance is provided.