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 anh ydrase 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 pr oteins with the lowest overall values of net charge (either positive or neg ative), 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) demonstra te a nonlinear correlation with n. Several observations made in this work s uggest that shifts in the values of pk(a) of the ionizable groups on these proteins likely contribute to the observed nonlinear correlation. Debye-Huc kel theory was used to calculate values of electrostatic potential at the s urface of the derivatives of all six proteins from the measured values of m u(electro). These values were plotted against the values of electrostatic p otential calculated by assigning a charge to each protein in direct proport ion to n. The data for all six proteins fell along a single common curve, r egardless of the concentration of monovalent cations in the electrophoresis buffer.