Immobilized urokinase column as part of a specific detection system for plasminogen species separated by...

Immobilized urokinase was used as part of a post-column reactor for the specific detection of human plasminogen species which were fractionated using a high-performance affinity column. After on-line activation of each peak, plasmin activity was measured by mixing the eluate with a specific fluorogenic substrate and the product was detected by a fluorescence monitor. This detection system gave linear calibration graphs for both purified plasminogens (0.1-50 micrograms) and plasminogens contained in plasma (25-100 microliters). Relative standard deviations for the determination of plasminogens in plasma were 6.1-6.6% (n = 12), showing good reproducibility. The detection limit was as low as 0.1 micrograms of plasminogen. Immobilized urokinase was very stable and no appreciable decrease in activity was found after 100 cycles of operation. In combination with an immobilized benzamidine column, this system made it possible to separate and detect Glu-plasminogen and Lys-plasminogen contained in human plasma samples as small as 100 microliters without any pretreatment.