Effects of urea induced protein conformational changes on ion exchange chromatographic behavior

Urea is widely employed to facilitate protein separations in ion exchange chromatography at various scales. In this work, five model proteins were used to examine the chromatographic effects of protein conformational changes induced by urea in ion exchange chromatography. Linear gradient experiments were carried out at various urea concentrations and the protein secondary and tertiary structures were evaluated by far UV CD and fluorescence measurements, respectively. The results indicated that chromatographic retention times were well correlated with structural changes and that they were more sensitive to tertiary structural change. Steric Mass Action (SMA) isotherm parameters were also examined and the results indicated that urea induced protein conformational changes could affect both the characteristic charge and equilibrium constants in these systems. Dynamic light scattering analysis of changes in protein size due to urea-induced unfolding indicated that the size of the protein was not correlated with SMA parameter changes. These results indicate that while urea-induced structural changes can have a marked effect on protein chromatographic behavior in IEX, this behavior can be quite complicated and protein specific. These differences in protein behavior may provide insight into how these partially unfolded proteins are interacting with the resin material.