Determination of Protein Charge in Aqueous Solution Using Electrophoretic Light Scattering: A Critical Investigation of the Theoretical Fundamentals and Experimental Methodologies.

Studies are reported of the measurement of electrophoretic mobilities of bovine serum albumin (BSA) in aqueous potassium chloride solutions as a function of ionic strength and pH using electrophoretic light scattering (ELS). It is demonstrated that the use of palladium or platinum electrodes should be avoided and that platinized platinum electrodes are necessary to avoid interference from unwanted electrochemical phenomena at the electrode-liquid interface. Potentiometric acid titration was performed to quantify the amount of protonic charge per protein molecule at the same pH values as the electrophoretic mobility measurements. It is shown that appropriate selection of an electrokinetic model yields excellent agreement between predicted and experimental electrophoretic mobilities across the ranges of pH and ionic strength studied in accordance with the protonic charge values obtained by titration. The experimental results are explained in terms of protonation, chloride counterion binding, and protein molecule permeability. This work highlights specific requirements of using ELS for confident analysis of proteins in aqueous solutions.