Heat-Induced Protein Oxidation as an Impediment for Thermal Stability Measurements: A Case Study on Cytochrome c.

Authors

Evelyn H MacKay-Barr, Lars Konermann

Published in

Analytical chemistry. Oct 06, 2025. Epub Oct 06, 2025.

Abstract

Thermal unfolding ("melting") experiments are widely used for protein stability measurements. These assays probe spectroscopic properties of the protein while gradually increasing the solution temperature until unfolding is complete. Thermodynamic parameters are obtained from fits of the resulting profiles. Differential scanning calorimetry relies on similar concepts. Meaningful stability measurements require reversible conditions, where the protein fluctuates between its native and unfolded states (N ⇌ U), as governed by the temperature-dependent equilibrium constant. A simple reversibility test is to ensure that heating and cooling (unfolding and refolding) profiles are superimposable. Unfortunately, such tests are not commonly performed. Here, we focused on cytochrome c, one of the most widely used model proteins for folding studies. Surprisingly, thermal unfolding/refolding was found to be irreversible, even in the absence of aggregation. Using mass spectrometry (MS), we traced the origin of this irreversibility to oxidative side chain modifications that accumulate during thermal assays. By gradually altering the covalent composition of the protein, oxidation creates a scenario far from ideal N ⇌ U conditions, rendering the validity of fitted thermodynamic parameters questionable. Oxidation at Tyr, Trp, and Met residues was promoted by dissolved O₂. It appears that the role of oxidation as an impediment for protein stability assays has been overlooked in the past. While the use of deoxygenated solutions represents a partial remedy, it is hoped that better oxidation suppression strategies will be developed in the future. In any case, it is advisable to perform MS measurements alongside thermal protein stability experiments to ensure that problems related to oxidation-mediated irreversibility are properly recognized.

PMID:
41052362
Bibliographic data and abstract were imported from PubMed on 07 Oct 2025.

Read full publication at:
Please sign in to see all details.

Stats

1-terrible, 9-excellent. How would you rate this publication? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.