Correlations between the charge of proteins and the number of ionizable groups they incorporate: Studies using protein charge ladders, capillary electrophoresis, and Debye-Huckel theory

The values of electrophoretic mobility, mu(electro), of bovine carbonic anhydrase II, human carbonic anhydrase II, cytochrome c, lysozyme, superoxide dismutase, ovalbumin, and derivatives of these proteins produced by partial neutralization of Lys epsilon-NH3+ and/or Asp and Glu carboxyl groups were measured using capillary electrophoresis (CE). For derivatives of these proteins with the lowest overall values of net charge (either positive or negative), the values of mu(electro) and the values of charge measured by CE, Z(CE), demonstrate a linear correlation with the number of charged groups, n, converted to neutral derivatives. For derivatives of these proteins with larger values of net charge, the values of mu(electro) and Z(CE) demonstrate a nonlinear correlation with n. Several observations made in this work suggest that shifts in the values of pk(a) of the ionizable groups on these proteins likely contribute to the observed nonlinear correlation. Debye-Huckel theory was used to calculate values of electrostatic potential at the surface of the derivatives of all six proteins from the measured values of mu(electro). These values were plotted against the values of electrostatic potential calculated by assigning a charge to each protein in direct proportion to n. The data for all six proteins fell along a single common curve, regardless of the concentration of monovalent cations in the electrophoresis buffer.