ENGINEERING SURFACE-CHARGES IN A SUBTILISIN - THE EFFECTS ON ELECTROPHORETIC AND ION-EXCHANGE BEHAVIOR

The introduction or removal of multiple charged amino acid residues in the subtilisin Savinase(TM) by genetic engineering allowed us to modi fy the electrostatic properties of this enzyme in a systematic way. Th e effects of these charge changes were investigated experimentally usi ng ion-exchange chromatography and electrophoretic mobility in native gels all under identical conditions. The experiments clearly demonstra ted that the overall charge of a given protein is not the only factor determining electrophoretic mobility at low or moderate ionic strength s. For a series of variants having identical overall positive charge a linear relation was observed between mobility towards the cathode and the total number of charged residues present. This effect was found t o depend on the type of (chloride) salt used: calcium ions give rise t o complete screening of all negative charges, whereas only partial scr eening is found for magnesium and sodium ions. In contrast, in the pre sence of sodium phosphate the overall charge of the enzyme becomes sli ghtly negative. These data indicate that cations as well as anions may strongly perturb the overall charge of proteins depending on the type of salt and on the number of charged amino acid residues present. The ion-exchange behaviour demonstrated similar results, i.e. showing str onger enzyme adsorption with increasing numbers of surface charges on a cation-exchange column run below the isoelectric point of the protei ns. However, the apparent sign reversal noted above for electrophoresi s with sodium phosphate did not appear in the ion-exchange experiments . This work provides further insight into the adsorption of proteins t o surfaces and the role played by small ions, particularly when electr ostatic forces dominate the adsorption process.