THE LOSS OF IN-VIVO ACTIVITY OF RECOMBINANT-HUMAN-ERYTHROPOIETIN BY ACTIVE OXYGEN SPECIES

The effects of active oxygen species on the in vivo activity of recomb inant human erythropoietin (EPO) treated by Fenton system, xanthine (X ) plus xanthine oxidase (XO) system and hydrogen peroxide (H2O2) has b een studied by means of counting the increase in number of hemolyser-r esistant cells (HRCs) in EPO-injected mice. The results showed that bo th Fenton and X plus XO systems caused a significant reduction of the activity in proportion to the concentration of generated active oxygen species. Meanwhile, the treatment of EPO with H2O2 alone resulted in a relatively slight reduction of the activity. Electrophoretic studies on the structure of EPO revealed that its main protein band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) disappeared in proporti on with the extent of exposure to active oxygen generating systems. Bo th Fenton and X plus XO systems caused a significant loss of fluoresce nce in the pyridylamino (PA-) sugar chain in proportion to the concent ration of generated active oxygen species, and no degradation products in the sugar chain part of the PA-sugar chain were detected. This sho wed that aromatic groups in EPO were sensitive to attack by active oxy gen species. These results provide evidence that hydroxyl radical and other active oxygen species have a potential to react with EPO, leadin g to a reduction of its in vivo activity.