Measurement of Specific Protease Activity Utilizing Fluorescence Polarization

Levine, Leanna; Michener, Marshall L.; Tóth, Máté; Holwerda, Barry C.

doi:10.1006/abio.1997.2047

Cited by 59 publications

(33 citation statements)

References 12 publications

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“…The most common methods for detecting degradative enzyme activity utilize fluorogenic and chromogenic substrates [3,[11][12][13][14][15], fluorescence resonant energy transferbased substrates [16,17], fluorescent polarization [18,19] and zymography [20][21][22][23]. While the final assay and detection steps for these substrates may be rapid and relatively sensitive, they all require that the initial blood sample be processed (e.g.…”

Section: Introductionmentioning

confidence: 99%

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

Lefkowitz

Marciniak

et al. 2010

Electrophoresis

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In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Charge-changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic alpha-chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both alpha-chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross-reactivity with the trypsin-like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin-specific), a detection limit of about 10-20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point-of-care diagnostics.

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Section: Introductionmentioning

confidence: 99%

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

Lefkowitz

Marciniak

et al. 2010

Electrophoresis

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show abstract

“…Protease activity is commonly measured using zymography [30][31][32][33], fluorogenic and chromogenic substrates [13,[34][35][36][37][38], fluorescence resonant energy transfer-based substrates [39,40], or with fluorescent polarization [41,42]. Although these methods are rapid and sensitive in samples that have been processed into plasma or serum and subsequently diluted, they cannot be used for direct measurement of protease activity in whole blood.…”

Section: Introductionmentioning

confidence: 99%

Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis

2010

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The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

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“…However, it has been reported that FP is generally not a good method for enzyme characterization except for those enzymes that have Km values below 10 μmol/L. 18 The FP signal increases with molecular weight, so the smaller peptide will have less FP signal. Thus, the kinetic parameters can not be accurately determined by FP method.…”

Section: Introductionmentioning

confidence: 99%

Capillary Electrophoresis with Laser-Induced Fluorescence Detection as a Tool for Enzyme Characterization and Inhibitor Screening

Liu

et al. 2008

ANAL. SCI.

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Measurement of Specific Protease Activity Utilizing Fluorescence Polarization

Cited by 59 publications

References 12 publications

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis

Capillary Electrophoresis with Laser-Induced Fluorescence Detection as a Tool for Enzyme Characterization and Inhibitor Screening

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