Development of a capillary electrophoresis method to monitor protein oxidation and antioxidant protection.

The development of a new capillary zone electrophoresis (CZE) method for the determination of protein oxidation by free radicals is described. The effects of various experimental conditions such as type of capillary. pH, buffer concentration, capillary dimensions, temperature. applied voltage, and addition of some organic solvents, were investigated. The sample injection procedure was also optimized. Target proteins investigated here (lysozyme, human serum albumin, and beta-lactoglobulin A) were effectively damaged by free radicals generated in the aqueous phase from 2,2'-azobis[2-amidinopropane] (AAPH). The new method allowed global protein fragmentation to be quantified as a function of time by monitoring the decrease in peak height of the native protein. Furthermore. the protective efficacy of antioxidants was assessed by concentration-inhibition curves against protein fragmentation. The method is thus suitable to screen compounds able to protect proteins against oxidative damag