Glutathione: A Tripeptide With One Bond in the Wrong Place
Glutathione is three amino acids long, which makes it a tripeptide by any reasonable definition. But one of its two bonds is not a peptide bond in the usual sense, and that single structural detail explains most of what is unusual about handling and analysing it.
The gamma linkage
Glutathione is gamma-Glu-Cys-Gly. In an ordinary peptide, each bond forms between the alpha-carboxyl of one residue and the alpha-amino group of the next. Here the glutamate contributes its side-chain carboxyl, the one on the gamma carbon, instead of its alpha-carboxyl.
The result is a molecule with a free alpha-amino group and a free alpha-carboxyl on the glutamate, both of which stick out from the chain rather than continuing it. Everything downstream follows from that arrangement.
Formula, mass and registry
Reduced glutathione is C10H17N3O6S, monoisotopic 307.08 Da, average 307.32 Da, CAS 70-18-8. It is usually abbreviated GSH, where the H denotes the free thiol.
The oxidised dimer, GSSG, is two molecules joined by a disulfide bond: C20H32N6O12S2, monoisotopic 612.15 Da, average 612.63 Da, CAS 27025-41-8. Two hydrogens are lost in forming the bond, which is why the dimer is 612.6 rather than 614.6.
Why standard peptide sequencing does not read it
Edman degradation removes residues one at a time from a free alpha-amino terminus, and the chemistry requires that the amine be alpha to the carbonyl that continues the chain. In glutathione the free alpha-amine is on a residue whose chain-continuing bond comes from the gamma position, so the reaction does not proceed as it would on a normal peptide.
The same logic applies to aminopeptidase enzymes, which is the biochemical reason the gamma linkage exists. For analytical purposes, the consequence is that identity is established by mass and by chromatographic comparison against a reference standard rather than by sequencing.
The thiol is the whole stability problem
The cysteine thiol oxidises in air, and the product is the disulfide-linked dimer. The reaction is catalysed by trace metals and accelerated at neutral and alkaline pH; it is slow in dry solid form and fast in aerated solution above pH 7.
This means the ratio of reduced to oxidised glutathione in a sample is a function of how it was stored and handled, not only of how it was made. A certificate reporting a GSH to GSSG ratio is reporting a snapshot, and a solution left standing at room temperature will not match it a week later.
Solutions prepared in slightly acidic water oxidise more slowly than those prepared in neutral buffer. Adding a chelator to sequester trace metals slows it further.
Chromatography of a very polar molecule
Glutathione carries a free amine, two carboxyls and a thiol on a ten-carbon skeleton. It is extremely polar and has almost no hydrophobic surface, so on a conventional C18 column it elutes at or near the void volume.
The methods that work are the same ones used for other small polar peptides: an aqueous-stable bonded phase run at very low organic content, ion-pairing chromatography, or hydrophilic interaction chromatography. GSSG is more retained than GSH under most of these conditions because the dimer is larger and presents more surface, which conveniently means the two separate.
Detection is the awkward part
There is no aromatic residue, so 280 nm is useless. At 214 nm the two amide bonds respond, but weakly for a molecule this small, and glutathione is often analysed at concentrations where that response is marginal.
Laboratories therefore commonly use one of three alternatives: electrochemical detection, which responds specifically to the free thiol and does not see the dimer; pre-column derivatisation with a thiol-reactive reagent that adds a chromophore or fluorophore; or mass spectrometric detection. Each answers a slightly different question, and a certificate should say which was used, because a thiol-specific method by construction reports nothing about the oxidised fraction.
Naming and forms
Glutathione appears as GSH, as reduced glutathione, as L-glutathione and simply as glutathione, which is ambiguous because the word alone does not distinguish reduced from oxidised. Salt forms exist, and so does S-acetyl glutathione, which is a distinct compound with an acetyl group on the sulfur and a mass 42 daltons higher.
A document should state the oxidation state and the salt form. Those two facts, with the mass, identify what is in the vial.
What confirms a lot
An accurate mass at 307.08 Da for the reduced form, a chromatogram from a method that retains it and separates it from the 612.6 dalton dimer, and a stated detection principle. For this compound the storage condition is part of the specification rather than a footnote to it.
Related reading
Salt form and counter-ion determine how much of a weighed quantity is the compound itself, which matters more for small molecules than for large ones. See counter-ions and salt form. For another catalogue compound that is a cofactor rather than a peptide, see NAD as a coenzyme in cellular energy research.
