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

The development of a new capillary zone electrophoresis (CZE) method for th e determination of protein oxidation by free radicals is described. The eff ects of various experimental conditions such as type of capillary. pH, buff er concentration, capillary dimensions, temperature. applied voltage, and a ddition of some organic solvents, were investigated. The sample injection p rocedure was also optimized. Target proteins investigated here (lysozyme, h uman serum albumin, and beta-lactoglobulin A) were effectively damaged by f ree radicals generated in the aqueous phase from 2,2'-azobis[2-amidinopropa ne] (AAPH). The new method allowed global protein fragmentation to be quant ified as a function of time by monitoring the decrease in peak height of th e 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 ag ainst oxidative damage.