ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 338 (2005) 151–158 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.039 Optical zymography for speciWc detection of urokinase plasminogen activator activity in biological samples Benedict Law a,1 , Jong-Kai Hsiao a,1,2 , Thomas H. Bugge b , Ralph Weissleder a , Ching-Hsuan Tung a,¤ a Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA b Proteases Tissue Remodeling Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institute of Health, Bethesda, MD 20892, USA Received 4 October 2004 Available online 13 December 2004 Abstract Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymo- graphic procedure that speciWcally detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA Xuorescent 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 buVer at pH 7.4 generates Xuo- rescence 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 plas- minogen and, therefore, can eYciently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demon- strated a highly correlative relationship (r 2 D 0.999) between the amount of uPA (over the range of 0.1–8.0 ng) and the average intensity of the Xuorescent band. We were able to directly measure uPA activities in diVerent cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.  2004 Elsevier Inc. All rights reserved. Keywords: Optical zymography; Urokinase; Fluorescence; Probe The involvement of urokinase plasminogen activator (uPA) 3 and matrix metalloproteinases (MMPs) in extra- cellular matrix degradation has been documented exten- sively over the past decades [1,2]. The degradation mechanism is considered to be a crucial step in tumor invasion [3–6]. Zymography is a routine electrophoretic technique to identify proteolytic activities in polyacryl- amide gels under nondenaturing conditions. It allows the estimation of the enzyme molecular weight and the iden- tiWcation of the enzyme of interest in a complex mixture of proteases. This method was originally applied to detect urokinase activity by using a gelatin substrate slab gel [7]. Since then, zymography with gelatin or casein has similarly been applied to detect other proteases [8,9]. The advantage to using zymography is the superior detection limit over other methods, including enzyme-linked immunosorbent assay (ELISA) and Western blots [10]. Despite the extensive applications, substrate speciWcity * Corresponding author. Fax: +1 617 726 5708. E-mail address: tung@helix.mgh.harvard.edu (C.-H. Tung). 1 These authors contributed equally. 2 Current address: Department of Medical Imaging, National Tai- wan University Hospital, Taipei, Taiwan. 3 Abbreviations used: uPA, urokinase-type plasminogen activator; MMP, matrix metalloproteinase; ELISA, enzyme-linked immunosor- bent assay; SDS, sodium dodecyl sulfate; PBS, phosphate-buVered saline; tPA, tissue plasminogen activator; TEMED, N,N,N',N'-tetra- methylethylenediamine; DMEM, Dulbecco’s modiWed Eagle’s medium; HBSS, Hanks’ balanced salt solution; EDTA, ethylenediamine tetraacetic acid; PAGE, polyacrylamide gel electrophoresis; HMW, high- molecular weight; LMW, low-molecular weight; NIR, near-infrared.