Development of a high-performance capillary isoelectric focusing technique with application to studies of microheterogeneity in chicken conalbumin

Abstract

A robust, simple, reproducible isoelectric focusing method using capillary electrophoresis that exhibits high stability, migration time reproducibility and pH linearity over a wide pH gradient was developed. Consecutive runs (over 113 runs) of several proteins and one peptide with isoelectric points (p Is) ranging from 9.45 to 2.75 yielded excellent migration time reproducibility (<2% R.S.D.). Experimental parameters including buffer aging and capillary-to-capillary variation were thoroughly examined and optimized to improve the migration time reproducibility. The capillary isoelectric focusing (CIEF) method was applied to the analysis of chicken conalbium (ovotransferrin), an iron-binding protein in egg white. Conalbium (low iron content) separated into three major components with p Is of 7.2, 6.6 and 6.2. When the protein was saturated with iron (2 Fe/mol), a shift to lower p Is was detected. Chicken serum transferrin subjected to CIEF gave a pattern similar to conalbium with three p Is of 7.1, 6.6 and 6.1, indicating that it was not fully saturated with iron. Thus, CIEF can be used as a potential analytical method to provide information about the metal-binding properties of specific metalloproteins.