RELATION OF PROTEIN ELECTROSTATICS COMPUTATIONS TO ION-EXCHANGE AND ELECTROPHORETIC BEHAVIOR

Although retention mechanisms in the various types of chromatography used for protein separations are understood qualitatively, methods have not yet been demonstrated for relating retention quantitatively to protein structure, surface structure, and solvent conditions. The mechanism most amenable to development of such techniques is electrostatic interactions, which are dominant in ion-exchange chromatography. In this work molecular electrostatics computations are performed on proteins using recently developed biophysical methods and applied to determining the mean potential over the molecular surface. These results are then used as the basis for comparison with ion-exchange retention times over a range of pH values. An excellent correlation, with no adjustable parameters, is found between the mean surface potential and retention time for one set of previously reported data, while the correlation between net charge and retention time is much poorer. A more quantitative comparison, between the mean surface potential of ribonuclease A and the zeta-potential evaluated from electrophoretic mobility data, is less successful; there are various possible explanations for the discrepancy. The approach proposed here, which treats the molecular electrostatics in quantitative detail, appears to be a promising one for use in explaining and predicting protein behavior in electrostatically controlled separations.