Cytochrome c unfolding on an anionic surface

• Published in: Journal of Chromatography A Vol. 863; no. 2; pp. 137 - 146
• Main Authors: Herbold, Craig W, Miller, John H, Goheen, Steven C
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 26-11-1999; Elsevier
• Subjects: Adsorption; Analytical, structural and metabolic biochemistry; Anions; Biological and medical sciences; Buffers; Cations; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cytochrome c Group - chemistry; Cytochromes; Fundamental and applied biological sciences. Psychology; Hemoproteins; Metalloproteins; Protein Folding; Proteins; Sodium Chloride; Solutions; Temperature; Time Factors; Proteins; Cytochromes; Ion-exchange chromatography; Cytochrome c; Separation method; Hemoprotein; Analysis method; Chromatographic retention; Unfolding; Temperature effect; Retention time; Conformational changes; Protein; Ion exchange chromatography; Ultraviolet visible spectrometry
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(99)00975-9

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°C, and a significant loss of cytochrome
c occurred between 55°C and 70°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.