Protease activity in human urine erythropoietin preparations

The presence of protease activity in 22 of 23 crude urine erythropoietin (Ep) preparations was identified by two different protease assays. Protease activity was enhanced at pH 4.5. Seven known protease inhibitors, two chelating agents, a sulfhydryl reagent and a chaotrophic agent failed to inhibit the protease activity of these Ep preparations. However, heating at 80 degrees C for 5 min uniformly eliminated protease activity without affecting Ep activity. In view of the sensitivity of cell surface proteins to proteolytic attack, the presence of protease activity in crude preparations of Ep should be considered when such preparations are employed in cell culture experiments.