Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples.
Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymographic procedure that specifically detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA fluorescent probe, which is copolymerized in sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis and renaturation, enzymatic digestions of the substrate in 50 mM of Tris buffer at pH 7.4 generates fluorescence emission at 695 nm. The enzymatic activities can be analyzed directly by conventional gel imaging systems with a detection limit of 40 pg. This protocol is fast (hours) and does not require staining and destaining steps. The procedure is independent of plasminogen and, therefore, can efficiently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demonstrated a highly correlative relationship (r2=0.999) between the amount of uPA (over the range of 0.1-8.0 ng) and the average intensity of the fluorescent band. We were able to directly measure uPA activities in different cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.
Law
Benedict
B
Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
Hsiao
Jong-Kai
JK
Bugge
Thomas H
TH
Weissleder
Ralph
R
Tung
Ching-Hsuan
CH
D002364:Caseins; D003470:Culture Media / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019073:HT29 Cells; D006801:Humans; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis*
D002364:Caseins; D003470:Culture Media; D014568:Urokinase-Type Plasminogen Activator