Figure 1.

Successive lysine modifications of rHuEPO. (A) Analysis of free amino acid loss by TNBS assay showed diverse yields of rHuEPO modification in various reactions. Based on these findings, the glutaraldehyde modification and guanidination were considered as the most efficient reactions. (B) Immunoblot of HEK-293 and P19 cells showed that both cell lines expressed EPOR and IL3RB. (C) LDH release from HEK-293 cells after induced oxidative cell injuries demonstrated that each modified EPO has different cytoprotective activities. The LDH release of H₂O₂-treated HEK-293 cells was 57.23%, which was significantly increased as compared with the non-treated cells and blank media (data not shown). Pretreatment with rHuEPO and modified EPOs significantly reduced LDH. (*P < 0.05 vs. control, ŧP < 0.05 vs. rHuEPO). (D) LDH release from P19 cells subjected to serum deprivation is an index of cell damage. The LDH release was highest in the control cells. Pretreatment with EPO derivatives significantly reduced LDH release, suggesting the cytoprotective properties of those molecules. (*P < 0.05 vs. control, ŧP < 0.05 vs. rHuEPO).