Stabilizing effect of poly(ethyleneimine) against metal catalyzed oxidation of proteins

BRECCIA JD,; ANDERSSON MM,; HATTI-KAUL R,

Toulouse

Conferencia; 3rd Internacional Conference on Protein Stabilisation; 2002

Metal catalyzed oxidation (MCO) of proteins has been recognized to be an important cause of inactivation of therapeutic proteins and peptides during bioprocessing and storage, and is also associated with tissue damage during various pathological states. Chelating agents are commonly used as additives to suppress the inactivation of proteins by MCO reactions. We have earlier demonstrated a cationic polymer, poly(ethyleneimine) (PEI) to be superior to several other additives as a stabilizer against oxidation of porcine lactate dehydrogenase (LDH). The protection provided by PEI of different molecular weights (2000, 25 000 and 2.6 x 106, respectively) has been further analysed in MCO systems containing Cu2+/Fe2+ ions and hydrogen peroxide. Comparisons were made with the chelators EDTA and Desferal, respectively. The analytical chelating capacity of PEI was found to be around 1 mole of Cu2+ per 10 moles of ethyleneimine. Oxidation was mainly monitored by way of its effect on protein aggregation. Aggregation of LDH with increasing [PEI monomer]/[metal ion] molar ratio followed a similar trend as with the other chelators, showing an initial enhancement and a subsequent decrease until it was undetectable. The LDH activity however showed a monotonic increase with increasing concentrations of the chelator. The concentration of PEI, especially of higher molecular weights, required for full protection of the enzyme was lower than that needed for complete chelation of metal ions. PEI was further shown to inhibit the formation of hydroxyl radicals generated in both copper and iron based MCO reactions in contrast to the other chelators which are more selective in their protective effect.