title: Cytochrome c unfolding on an anionic surface.
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.
authors.affiliation: Department of Chemical Sciences, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: C W
authors.initials: CW
authors.lastname: Herbold
authors.forename: J H
authors.initials: JH
authors.lastname: Miller
authors.forename: S C
authors.initials: SC
authors.lastname: Goheen
mesh_terms: D000327:Adsorption; D000838:Anions; D002021:Buffers; D002412:Cations; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000737:chemistry*; D017510:Protein Folding; D012965:Sodium Chloride; D012996:Solutions; D013696:Temperature; D013997:Time Factors
chemical_list: D000838:Anions; D002021:Buffers; D002412:Cations; D003574:Cytochrome c Group; D012996:Solutions; D012965:Sodium Chloride