Why does dimerisation accelerate at 2–8 °C rather than proceeding linearly?

Setup, so nobody has to ask: dimerisation · 2–8 °C.

The empirical answer seems settled. The explanation does not.

If the honest answer is that nobody knows, I would rather hear that than a plausible story.

Is the standard explanation correct, and if so, what is the evidence for it?

Because temperature enters the rate constant through an exponential, so equal steps in temperature multiply the rate instead of adding to it. Arrhenius puts the rate proportional to exp(−Ea/RT); the working approximation is a doubling per 10 K, which takes 5, 15, 25 and 35 °C to multipliers of 1, 2, 4 and 8. The steps in temperature are equal and the steps in rate are not, and that is the whole of the observation. At 2–8 °C the same rule gives about 1 times the refrigerated rate, and another 10 K would roughly double it again. Two chains join, usually through a disulfide, so the product is roughly twice the mass and shows up as a late peak — or as nothing, if it never comes off the column. Ea differs by route, so the ranking of routes changes with temperature too — which is why accelerated data extrapolates badly and why nobody should read a 40 °C study as a fast version of a 5 °C one.

Asparagine and glutamine are the deamidation risk, and methionine is the oxidation risk.

Oxidation targets methionine, cysteine and tryptophan, adding sixteen daltons per oxygen. It is catalysed by trace metals and promoted by dissolved oxygen and by light.

Specifically, light exposure matters for tryptophan-containing sequences and for anything with a chromophore. Amber vials and a closed box are free mitigations.

Apparent loss in a dilute preparation is usually adsorption rather than degradation and is worth ruling out first.

Swirl, never shake. Aggregation is a handling problem more than a time problem.

The honest answer is that most reported "degradation" is adsorption and dilution error rather than chemistry.

Aggregation is physical: peptides unfold at air-liquid interfaces and associate. Shaking maximises that interface, which is why swirling and shaking produce visibly different outcomes on the same vial.

A mass spectrum resolves most of this: minus eighteen is dehydration or succinimide, plus one is deamidation, plus sixteen is oxidation, and an unchanged mass with a shifted retention time is an isomer.

Metal-catalysed oxidation of methionine is documented across peptide and protein formulations and is why chelators appear in some formulations.

Sequence decides which pathways are even available. Check the residues.

edited 16 Aug 2026 by gradient_slope — fixed an arithmetic slip in the third paragraph

This is answerable from the chemistry rather than from anecdote, which is unusual and welcome.

Deamidation converts asparagine or glutamine to the corresponding acid via a succinimide intermediate, adding one dalton. It is base-catalysed, accelerates above neutral pH and is the dominant aqueous pathway for many peptides.

It helps to be literal here: adsorption onto glass and plastic is significant at low concentrations — micrograms per millilitre — and negligible at milligrams per millilitre. It is the usual explanation for an apparent loss in a dilute preparation.

At dilute concentrations, suspect adsorption before you suspect chemistry.

The relevant point is that a mass shift of plus one dalton is deamidation and plus sixteen is oxidation, so degradation is often visible in a mass spectrum if anyone looks.

Freeze-thaw cycling drives aggregation through concentration at the ice interface and pH shifts as buffer components crystallise out at different rates. Each cycle costs something.

The caveat is that none of these pathways can be seen by looking at a vial, and a clear solution can be substantially degraded.

Cold, dry, dark, still. Those four words cover most of the mitigation.

Stated carefully, aggregation is a physical process and is the one most often caused by handling rather than by time.

Hydrolysis cleaves the backbone, most readily at aspartate-proline and aspartate-glycine sequences, and is acid-catalysed. In a dry solid it barely proceeds at all.

Nothing here is medical advice, and research-use compounds are not approved for human use.

A mass spectrum names the pathway. Plus one, plus sixteen, minus eighteen.