Significance of Charge Regulation in the Analysis of Protein Charge Ladders

Analysis of protein charge ladders using capillary electrophoresis (CE) provides a method of determining charges of proteins. This method has disregarded the effect of charge compensationa response of the protein and its environment to a change in electrostatic potential on the surface of the protein. This work examines the difference in charge, ΔZ, between the first two rungs of the ladder of bovine carbonic anhydrase II (BCAII) as a function of pH and ionic strength using CE. These data were analyzed in three ways: using models based on Hückel theory and on charge regulation, and using linear regression. These analyses were in only qualitative agreement, and the differences between them suggest that simple theoretical models for the behavior of colloidal particles cannot establish the value of ΔZ accurately in proteins. Linear regression of mobilities of the rungs of charge laddersa method proposed in earlier workcontinues to be a computationally convenient method of estimating the charge Z0 of native proteins, but the accuracy of this method depends on the value of ΔZ. The absolute value of ΔZ cannot presently be established accurately. In the case of BCAII, we suggest ΔZ = −0.93 for the difference in charge between the first two rungs of the charge ladder at pH = 8.4 and 10 mM ionic strength. An estimate of the uncertainty in this value for BCAII due to uncertainties in the values of pKa of amino acids and of the hydrodynamic radius is ±0.02. Other uncertainties not considered in this analysis will make this value larger.