Effect of active oxygen radicals on protein and carbohydrate moieties of recombinant human erythropoietin

Our previous study showed that active oxygen radicals generated from a Fenton system and a xanthine plus xanthine oxidase system caused serious loss of in vivo bioactivity of recombinant human erythropoietin (EPO), a highly glycosylated protein. In the present study, we characterized the oxidative modifications to the protein and carbohydrate moiety of EPO, which lead to a reduction of its bioactivity. In vitro bioactivity was reduced when EPO was treated with oxygen radicals generated from a Fenton system in the presence of 0.016 mM H2O2, and the reduction was directly proportional to the loss of in vivo bioactivity. SDS-PAGE analysis showed that dimer formation and degradation was observed under more severe conditions (Fenton reaction with 0.16 mM H2O2). The tryptophan destruction was detected at 0.016 mM H2O2 and well correlated with the loss of in vitro bioactivity, whereas loss of other amino acids were occurred under more severe conditions. Treatment with the Fenton system did not result in any specific damage on the carbohydrate moiety of EPO, except a reduction of sialic acid content under severe condition. These results suggest that active oxygen radicals mainly react with the protein moiety rather than the carbohydrate moiety of EPO. Destruction of tryptophan residues is the most sensitive marker of oxidative damage to EPO, suggesting the importance of tryptophan in the active EPO structure. Deglycosylation of EPO caused an increased of susceptibility to oxygen radicals compared to intact EPO. The role of oligosaccharides in EPO may be to protect the protein structure from active oxygen radicals.