Objective To investigate the effect of glycosylation at Asn302 of pro-urokinase （pro-UK） on the stability in culture supernatant. Methods Nonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302.The pro-UK mutant and native pro-UK were transfected into dhfr

- -CHO cells

and serum-free culture supernatant was harvested and incubated at 4℃ and 37℃

respectively. The pro-UK activity in culture supernatant was measured by the optical density （OD） increase with time （12 hours） at 405 nm. Without thermolysin activation

the percentage of single chain pro-UK was measured. Results After 48 hours of incubation at 4℃

the activities of pro-UK mutant and native pro-UK decreased 3.7% and 2.9% respectively

and at 37℃ decreased 37.9% and 23.5%

respectively. The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37℃. The single-chain percentage of native pro-UK was higher than that of nonglycosylated mutant at both 4℃ and 37℃. Conclusion Higher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supernatant.