Oxidation and reduction: oxidising and reducing agents in the lab

Oxidation is the loss of electrons and reduction is the gain of electrons; because electrons have to go somewhere, the two always happen together, and the pair is called a redox reaction. An oxidising agent takes electrons and is itself reduced; a reducing agent gives electrons and is itself oxidised. This guide explains both, how to tell which is which, which parts of a peptide are most at risk of oxidation, and how common reducing agents such as DTT and TCEP are used.

Terms are defined in the glossary.

What oxidation and reduction mean

The simplest definitions are about electrons:

• Oxidation is the loss of electrons.
• Reduction is the gain of electrons.

A common memory aid is OIL RIG: oxidation is loss, reduction is gain.

Electrons can't simply disappear, so whenever one substance is oxidised, another is reduced. That pairing is a redox reaction.

In organic chemistry it is often easier to spot oxidation by what happens to the atoms:

Both views describe the same change. When methionine gains an oxygen atom, its sulfur gives up electron density to that oxygen and is oxidised. When two cysteine thiols (–SH) lose a hydrogen atom each and join as a disulfide (–S–S–), both sulfurs are oxidised.

What is reduction in chemistry?

Reduction is the gain of electrons. In organic chemistry it usually shows up as gaining hydrogen or losing oxygen. It always happens alongside an oxidation. In peptide work, the most common reduction is breaking a disulfide bond back into two thiols.

Oxidation states

An oxidation state (or oxidation number) is a way of tracking electrons: the charge an atom would have if each shared pair of electrons were given to the more electronegative atom. The main rules are (OpenStax):

• An atom in a pure element is 0.
• A single-atom ion has its own charge, such as +1 for Na⁺.
• Oxygen is usually −2, and hydrogen bonded to a non-metal is usually +1.
• The oxidation states in a molecule add up to its overall charge.

Sulfur shows how this tracks peptide chemistry:

Each step down the table is an oxidation, because the sulfur's oxidation state goes up.

How to identify the oxidising and reducing agent

Work out the oxidation state of each element before and after. The one that goes up has been oxidised, so that substance is the reducing agent. The one that goes down has been reduced, so that substance is the oxidising agent. The names feel back to front at first: a reducing agent is the thing that gets oxidised.

Worked example: zinc and copper sulfate. Zinc metal dropped into copper sulfate solution gives copper metal and zinc sulfate:

Zn(s) + Cu²⁺(aq) → Zn²⁺(aq) + Cu(s)

Two electrons pass from each zinc atom to each copper ion. Splitting that into half-equations makes the electrons visible: Zn → Zn²⁺ + 2e⁻ and Cu²⁺ + 2e⁻ → Cu.

Worked example: iodine and a thiol. When iodine joins two cysteine thiols into a disulfide:

2 R–SH + I₂ → R–S–S–R + 2 HI

Each sulfur goes from −2 to −1, so the thiol is oxidised and is the reducing agent. Iodine goes from 0 to −1 in the iodide, so it is reduced and is the oxidising agent. For the balanced-equation method, see balancing chemical equations.

Oxidation and reduction in peptides

Which residues oxidise

Most of a peptide's backbone is stable to oxidation under normal storage. The weak points are a few side chains:

These changes show up as new peaks on HPLC and as mass shifts in mass spectrometry. See reading impurity peaks for how to recognise them. For more on cysteine and its thiol group, see the 20 amino acids.

What drives it

Oxidation needs an oxidising agent. In a vial or a solution, the usual ones are:

• Oxygen from the air, especially in solution and at higher pH. How fast it acts depends on temperature and concentration like any other reaction; see rates of reaction.
• Trace metal ions, such as iron and copper, which speed up oxidation by oxygen even at very low levels (Stadtman, Free Radic. Biol. Med. 1990). See EDTA and chelating agents for how labs tie them up.
• Peroxides, which build up in some solvents and additives as they age. Ethers such as tetrahydrofuran and diethyl ether, and polymers such as polyethylene glycol and polysorbates, are known sources.
• Light, which can drive the oxidation of tryptophan and other aromatic residues.
• Dimethyl sulfoxide (DMSO), which can oxidise methionine and cysteine. The solubility guide suggests other organic solvents for those sequences.

Dry, cold, dark and sealed storage limits all of these. See how to store freeze-dried peptides.

Oxidising agents

An oxidising agent (or oxidant) takes electrons from something else, and is itself reduced in the process. Common laboratory oxidising agents include:

Strong oxidising agents carry the "flame over circle" hazard pictogram, because they can make other materials burn more fiercely. Keep them apart from flammable solvents. See COSHH and safety data sheets.

Reducing agents

A reducing agent (or reductant) gives electrons to something else, and is itself oxidised. In peptide and protein work, reducing agents are used mainly to break disulfide bonds and keep cysteine thiols in the free –SH form.

DTT was introduced by W. W. Cleland in 1964 and remains the standard thiol reductant (Cleland, Biochemistry 1964). A side-by-side study found the choice depends on the job (Getz et al., Anal. Biochem. 1999):

• Labelling. DTT blocked maleimide labels from attaching to thiols and had to be removed first. Labelling worked in the presence of TCEP, though less efficiently than with no reductant.
• Metal ions. Trace nickel ions oxidised DTT rapidly but didn't affect TCEP.
• Keeping solutions. TCEP lasted longer in buffers without a metal-binding agent. DTT lasted longer when one was present. See EDTA and chelating agents.

What reducing agents can and can't undo

• Disulfides are reversed easily. That is why SDS-PAGE samples include DTT or β-mercaptoethanol. See gel electrophoresis and SDS-PAGE.
• Methionine sulfoxide is not reversed by DTT, TCEP or β-mercaptoethanol under normal conditions. Once a methionine has oxidised, it stays oxidised in the vial.
• Tryptophan oxidation is not reversible either.

So the practical aim is to prevent oxidation, and to use reducing agents only for the reversible disulfide case.

Measuring free thiols

Ellman's reagent (DTNB) reacts with free thiols to release a yellow product that absorbs at 412 nm (Riddles et al., Methods Enzymol. 1983). Measuring that colour gives the number of free –SH groups in a sample. It shows whether cysteines are reduced or have formed disulfides. For how absorbance is turned into a concentration, see measuring peptide concentration.

Deliberate oxidation in synthesis

Not all oxidation is unwanted. Many peptides contain intended disulfide bonds that hold them in a ring. After the linear chain is made, the cysteines are joined on purpose by controlled oxidation, using air at a dilute concentration, iodine, or a mild oxidant. The disulfide then appears in the sequence notation as part of the structure, not as an impurity. See solid-phase peptide synthesis.

Related guides

For how oxidation fits among other breakdown routes, see shelf life and stability. For the functional groups that oxidise, see functional groups in peptides. For what an ion's charge means, see ions: cations, anions and charge.