Microcalorimetric Analysis of the Adsorption of Lysozyme and Cytochrome c onto Cation-Exchange Chromatography Resins: Influence of Temperature on Retention.

We studied the adsorption mechanism of two basic proteins, equine cytochrome c (Cyt) and chicken egg-white lysozyme (Lys), adsorbing onto negatively charged chromatography surfaces. In liquid chromatography, the retention volume of Lys was larger than that of Cyt on negatively charged ion-exchange resins. When the temperature increased, the retention volume of Cyt increased, whereas that of Lys clearly decreased. Both Lys and Cyt share similar physical characteristics, so the opposite behavior with increasing temperatures was surprising, indicating a more complex mechanism of adsorption may be involved. We analyzed the adsorption of these proteins by using isothermal titration calorimetry (ITC). The change in adsorption enthalpy determined by ITC allowed the understanding of the reason for and underlying driving forces of protein adsorption that resulted in this opposite behavior. Large exothermic enthalpies of adsorption were observed for Lys (-43.95 kJ/mol), and Lys adsorption was found to be enthalpically driven. On the other hand, endothermic enthalpies were dominant for Cyt adsorption (32.41 kJ/mol), which was entropically driven. These results indicate that dehydration and release of counterions play a more important role in Cyt adsorption and ionic interaction and hydrogen bridges are more significant in Lys adsorption. Understanding of the adsorption mechanism of proteins onto chromatography resins is essential for modeling and developing new, efficient chromatographic processes.