Effects of catholytes on the mobilization of proteins after capillary isoelectric focusing.

Purified proteins and human plasma proteins were separated by capillary isoelectric focusing and detected by cathodic mobilization, e.g. by replacing the cathodic sodium hydroxide solution by solutions containing another anion. The effect of catholyte anions on the resolution of mobilized proteins was examined. Compared with the chloride or phosphate anion, organic anions having small dissociation constants improved protein resolution, especially in the range of acidic proteins. Among the catholytes examined, acetic acid gave the best resolution for purified proteins and human plasma proteins. Measurement of the changes of electric current during the mobilization process indicated that organic anions with low mobility move slowly towards the anode and retard changes of pH gradient in the capillary. Isoelectric focusing, cathodic mobilization, and direct pH measurement in polyacrylamide gel columns (1.3 mm internal diameter and 38 mm long) also revealed these effects of low mobility anions. In order to visualize the behavior of human plasma proteins in the mobilization step, micro two-dimensional polyacrylamide gel electrophoresis was employed. The results indicated that the proteins migrate towards the cathode, preserving their relative positions attained during the isoelectric focusing step.