Antibody transformation by peroxynitrite as determined using capillary electrophoresis: a feasibility study

Peroxynitrite may be a physiologically relevant endogenous neurotoxin that forms following CNS trauma when excessive levels of NO and .O2 accumulate. Recently, peroxynitrite was found to inactivate the polyclonal antibody to cAMP. A feasibility study was performed to evaluate the use of capillary electrophoresis as an effective tool regarding the structural transformation of antibody following exposure to peroxynitrite with or without co-incubation with a peroxynitrite scavenger. A polyclonal antibody to cAMP and a monoclonal antibody to plasminogen activation inhibitor-1 were exposed to peroxynitrite with or without penicillamine coincubation. Samples were analyzed by an Applied Biosystems analytical capillary electrophoresis system, model 270A. Initial examination of the peroxynitrite scavenger penicillamine and its reaction with peroxynitrite showed a penicillamine migration peak at about 9.1 min and a presumed s-nitro adduct of penicillamine that migrated at 10.9 min. Exposure of either antibody to peroxynitrite resulted in structural transformation of protein based on changes in migration patterns. In addition, co-incubation with penicillamine prevented this transformation and preserved the pre-peroxynitrite migration patterns of antibodies. In cases of antibody reaction, s-nitro adduct formation could be simultaneously monitored. We found capillary electrophoresis to be ideally suited to this type of analysis. With capillary electrophoresis, we were able to simultaneously monitor the effects of peroxynitrite on large proteins and a small scavenger molecule. As a result, a complete record of the reaction was obtained within a single 15-min analysis period.