Cytochrome c unfolding on an anionic surface

It is now well accepted that the adsorption of proteins to solid supports s ometimes involves surface-mediated unfolding. A detailed understanding of t he adsorption and surface-mediated unfolding process is lacking. We selecte d a well studied protein, horse heart cytochrome c, and a weakly ionic supp ort 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 unfoldin g. Samples of cytochrome c were introduced to an anionic support, and a NaC l gradient was used to desorb the protein at different times and temperatur es. The profiles and retention times were monitored to examine the adhesive properties of cytochrome c to the anionic support. We found that protein r etention increased with time at temperatures as low as 0 degrees C, and a s ignificant loss of cytochrome c occurred between 55 degrees C and 70 degree s C. The loss of recovery of cytochrome c indicates irreversible surface-me diated unfolding. The changes in retention time may indicate more subtle tr ansitions, including reversible surface-mediated unfolding of cytochrome c. These results suggest that perturbations in the structure as well as unfol ding of cytochrome c can be detected at a lower temperature on an anionic s urface than in solution thereby acting like a catalyst for protein unfoldin g. (C) 1999 Elsevier Science B.V. All rights reserved.