Effects of urea-thermal denaturation on the high-performance cation-exchange chromatography of...

Retention parameters of alpha-chymotrypsinogen-A were determined by isocratic elution for a series of concentrations of calcium acetate and sodium acetate both in the presence and absence of urea. Under non-denaturing conditions of temperature and urea concentration, urea facilitated elution. Under reversible denaturing conditions a sharp drop in chromatographic retention was observed over a narrow temperature range which could be correlated with equilibrium measurements of the protein fluorescence. Retention of both native and denatured protein could be fit to a non-mechanistic retention model by plotting the log k' against log salt concentration. Conventional interpretation of these findings indicates that, while the number of ions displaced during binding is greater for the denatured protein, the affinity per ion decreases since the retention of denatured protein is much less than native. Elution profiles obtained under partially denaturing conditions showed a strong flow-rate dependence. We attribute these observations to a rate of equilibration between native and denatured protein that is on the timescale of the chromatographic rate processes.