Protein retention in ion-exchange chromatography: effect of net charge and charge distribution

The charge regulated slab model is used to evaluate the salt dependence of the retention of Staphylococcal nuclease A and its mutants in cation-exchange chromatography. An important feature of this work is that the net charge of the proteins is varied in two different ways: (a) by changing the eluent pH so that the charges are created by protonation and (b) by point mutation at position 116. Since the structure of Staphylococcal nuclease and the mutants are known, the pH dependence of retention data of the different mutants gives detailed insights into the retention mechanism. Experimental results show that the salt dependence of retention is affected more strongly by changes of the eluent pH than by point mutations. This implies that the amino acid in position 116 has only a moderately strong interaction with the stationary phase surface and that a patch on one side of the protein surface is mainly responsible for the electrostatic interaction with the surface.