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Updated: Aug 12, 2026

Surface Passivation for Single-molecule Protein Studies
Published on: April 24, 2014
Cytochrome c unfolding on an anionic surface
C W Herbold1, J H Miller, S C Goheen
1Department of Chemical Sciences, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Abstract:
It is now well accepted that the adsorption of proteins to solid supports sometimes involves surface-mediated unfolding. A detailed understanding of the adsorption and surface-mediated unfolding process is lacking. We selected a well studied protein, horse heart cytochrome c, and a weakly ionic support to examine some of the characteristics of protein adsorption under near-physiological conditions. We used high-performance liquid chromatography (HPLC) to investigate the effect of temperature on surface-mediated unfolding. Samples of cytochrome c were introduced to an anionic support, and a NaCl gradient was used to desorb the protein at different times and temperatures. The profiles and retention times were monitored to examine the adhesive properties of cytochrome c to the anionic support. We found that protein retention increased with time at temperatures as low as 0 degrees C, and a significant loss of cytochrome c occurred between 55 degrees C and 70 degrees C. The loss of recovery of cytochrome c indicates irreversible surface-mediated unfolding. The changes in retention time may indicate more subtle transitions, including reversible surface-mediated unfolding of cytochrome c. These results suggest that perturbations in the structure as well as unfolding of cytochrome c can be detected at a lower temperature on an anionic surface than in solution thereby acting like a catalyst for protein unfolding.
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