Determination of effective protein charge by capillary electrophoresis: Effects of charge regulation in the analysis of charge ladders

Protein charge ladders are an effective tool for measuring protein-charge a nd studying electrostatic interactions. However, previous analyses have neg lected the effects of charge regulation, the alteration in the extent of am ino acid ionization associated with differences between the pH at the prote in surface and in the bulk solution. Experimental:data were obtained with c harge ladders constructed from bovine carbonic anhydrase, The protein charg e for each element in the ladder was calculated from the protein electropho retic mobility as measured by capillary electrophoresis using the hindrance factor for a hard sphere with equivalent hydrodynamic radius, The protein charge was also evaluated theoretically from the amino acid sequence by ass uming a Boltzmann distribution in the hydrogen ion concentration. The calcu lations were in excellent agreement with the data, demonstrating the import ance of charge regulation on the net protein charge. These results have imp ortant implications for the use of charge ladders to evaluate effective pro tein charge in solution.