Glutathione is sold in its reduced form and spends every moment in solution trying to leave it. The molecule's entire function is to give up an electron, and it will do so to dissolved oxygen, to trace metal ions and to another glutathione molecule just as readily as to anything an experiment intends. A solution that was fully reduced when made can be substantially oxidised within a day, without any visible change. Understanding why is the difference between measuring glutathione and measuring what it turned into.
What the molecule is
A tripeptide: γ-glutamyl-cysteinyl-glycine. The first bond is unusual — glutamate is joined through its side-chain carboxyl rather than its backbone one, which is why ordinary peptidases do not cleave it and why it is not, strictly, a standard peptide bond. The working part is the cysteine. Its thiol group is the electron donor, and the whole redox chemistry of the molecule happens there.
Two glutathione molecules that each donate an electron end up joined through their sulphurs, forming the disulphide dimer GSSG. That is the oxidised form. In a cell, an enzyme recycles GSSG back to two reduced molecules; in a tube, nothing does.
| GSH (reduced) | GSSG (oxidised) | |
|---|---|---|
| Structure | One tripeptide, free thiol | Two tripeptides, disulphide-linked |
| Mass | 307 Da | 613 Da (two GSH minus two H) |
| Redox role | Electron donor | Spent |
| Appearance in solution | Colourless | Colourless |
| Separable by HPLC | Yes, but they are not the same peak | |
The fourth row is the practical problem. Nothing about a glutathione solution tells you which form it is in, and a solution that has half-converted looks exactly like one that has not.
What actually oxidises it in a vial?
Three things, usually together. Dissolved oxygen, which every aqueous solution holds unless it has been degassed. Trace transition metals — copper and iron at the parts-per-billion level found in ordinary buffers and glassware — which catalyse the reaction between thiol and oxygen so effectively that removing them slows oxidation by an order of magnitude. And pH: the thiol is far more reactive as the thiolate anion, which predominates above roughly pH 8, so a neutral or basic buffer oxidises glutathione much faster than a slightly acidic one.
How fast does it happen?
Fast enough to matter within a working day and slow enough to be invisible. In an ordinary neutral buffer at room temperature, open to air, a meaningful fraction can convert to GSSG in hours; refrigerated and slightly acidic, that stretches to days. Frozen, it largely stops. The rate is so sensitive to metal contamination that two laboratories following the same protocol can see very different stability, which is one reason published figures disagree. What does not happen is a plateau: the reaction runs until the thiol is gone.
Keeping it reduced
The measures follow from the causes, and the first two cost nothing.
- Slightly acidic solvent. Around pH 3–5 keeps the thiol protonated and slow. This is the single most effective step.
- Make it fresh. A solution prepared the day it is used has not had time to oxidise. Stocks should be aliquoted and frozen, not refrigerated for weeks.
- Chelate the metals. A small amount of EDTA in the buffer sequesters the catalytic copper and iron. This is incompatible with anything that itself needs those metals — a copper complex, for instance — so it is a choice rather than a default.
- Degas, or work under nitrogen. Removes the oxidant. More effort; worth it for anything quantitative.
- Keep it cold and dark. Light accelerates metal-catalysed oxidation; cold slows everything.
Does the powder oxidise too?
Slowly, and mainly if it takes up water. Dry reduced glutathione is reasonably stable sealed and cold; it is the dissolved thiol that is reactive. The usual precaution against moisture applies with extra force here — let a cold vial reach room temperature before opening it, because condensation inside a vial of glutathione starts the same oxidation chemistry in the solid. A certificate for reduced glutathione should report the reduced fraction, not just purity, since GSSG is a legitimate-looking impurity on a purity trace that treats both forms as glutathione.
Can oxidised glutathione be reduced back?
In a tube, only by adding a stronger reducing agent, which then contaminates the solution with something that has the same problem plus its own. Dithiothreitol will regenerate GSH from GSSG but leaves oxidised DTT behind and interferes with most downstream assays; tris(2-carboxyethyl)phosphine is cleaner but still an additive. Neither restores a solution to the state it would have been in if made fresh. For research purposes the answer is the same as for any degraded peptide solution: making it again costs less than rescuing it.
Why does this matter beyond glutathione itself?
Because glutathione is the model for every free thiol in the catalogue. Any peptide with an unpaired cysteine does the same chemistry — oxidises to a disulphide dimer, catalysed by metals, faster at high pH — and glutathione is simply the case where the whole molecule is that thiol. What is learned handling it transfers directly to why cysteine-containing peptides dimerise in a stock, and to why a free thiol is the first thing that rules a peptide out of a blend. It is the textbook case because it is the pure case.
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