Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model (original) (raw)
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BioTechniques, 2011
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Current Protocols in Chemical Biology, 2009
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Analytical Biochemistry, 1997
substrates offers an alternative method that avoids in-A fluorescence polarization assay was designed to terference, but disposal and safety concerns make this measure proteolytic cleavage of a specific peptide subapproach less attractive. strate for human cytomegalovirus protease. The pep-We present an assay for site-specific proteolytic actide substrate was derivatized by biotinylation of a tivity utilizing fluorescence polarization. The substrate g-aminobutyric acid-modified amino-terminus and lais a protease-specific peptide derivatized by biotinylabeled with 5-(4,6-dichlorotriazinyl)aminofluorescein tion of the amino-terminus and coupling of a fluoroat the carboxy-terminus. Incubation of this substrate phore at the carboxy-terminal end. Proteolytic activity with recombinant human cytomegalovirus protease was quantitated from the total fluorescence polarizaand subsequent addition of egg white avidin produced tion of the mixture of cleaved and uncleaved peptide a polarization signal that was proportional to the relaafter incubation with the protease. Since the fluorestive amounts of cleaved and uncleaved substrate. The cence polarization value is the ratio of orthogonal fluouncleaved substrate produced a high polarization rescence intensities, it is not sensitive to absorptive value upon binding to avidin, whereas the cleaved, interferants. We demonstrate the assay robustness to low-molecular-weight fluorescently tagged peptide absorptive interferants by measuring the polarization that cannot bind to avidin produced a low polarization of a constant concentration of biotin-fluorescein, biovalue. The inhibitory activity of a 3,4-dichloroisocoumtin, and avidin in the presence of increasing concentraarin against the protease was evaluated by comparing tions of dyes that absorb where fluorescein either abthe change in polarization with a noninhibited consorbs or emits. trol. The fluorescence polarization protease assay does not suffer from interference due to the presence of absorptive interferants making this a convenient, ho-MATERIALS AND METHODS mogenous assay for high throughput screening. ᭧ 1997 Chemicals and Reagents Academic Press Avidin, biotin-fluorescein, and 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) 1 were purchased from Molecular Probes (Eugene, OR). Bovine serum albumin The activity of proteases recognizing specific cleav-(fraction V; BSA), 3-[(3-chloramidopropyl)-dimethyage sites is usually measured using substrates conlammonio]-1-propane sulfonate (Chaps), g-aminobusisting of a specific peptide modified by the addition of tyric acid (Abu), 3,4-dichloroisocoumarin, mordant blue latently colorimetric or fluorescent moieties (1-6). The 3, eosin B, and biotin were purchased from Sigma (St. hydrolysis products of these synthetic substrates pos-Louis, MO). All other chemicals were analytical grade. sess spectral features that enable quantitative deter-Buffers were stored at 4ЊC after preparation in ultramination of substrate cleavage. Substrates of this type pure water (Millipore Milli-Q) and further filtered are widely used in the characterization of many differthrough 0.2-mm filters. Recombinant human cytomegaent proteases. However, evaluation of potential inhibitors in complex mixtures such as natural products ex-1 Abbreviations used: DTAF, 5-(4,6-dichlorotriazinyl)aminofluortracts can be severely limited because these mixtures escein; BSA, bovine serum albumin; Chaps, 3-[(3-chloramidopropyl)often contain other components that interfere with dimethylammonio]-1-propane sulfonate; PBS, phosphate-buffered saline; HCMV, human cytomegalovirus; Abu, g-aminobutyric acid.
Scientific Reports, 2017
Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/ Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/ specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. The irreversible peptide bond hydrolysis of proteins and polypeptides is the most conserved post-translational modification occurring in biochemical pathways in all living organisms 1,2. This reaction is catalyzed by proteases, which specifically recognize protein targets to control numerous significant biological processes, including cell survival and cell death and the immune response to various pathogens 3. The selectivity of proteases for binding and subsequently hydrolyzing a selected group of peptides or proteins is termed substrate specificity 4,5. The increasing number of chemical tools for substrate specificity profiling allows the development of new, more efficient and more selective small molecule substrates 6,7 , inhibitors 8 , and chemical probes 9 , which are useful for the determination of protease activity and the dissection of their physiological functions. Internally quenched fluorescent (IQF) peptide substrates constitute a convenient tool for examining the specificity of the largest group of proteases-endopeptidases 10. These substrates contain a paired fluorophore (donor) and quencher (acceptor), which are located on opposite sides of the scissile peptide bond 11,12. If the fluorophore
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Analytical Biochemistry, 1997
. The drawback of these assays is that proteases sometimes behave differently toward small peptides or David A. Menges, Damian L. Ternullo, in hydrolysis of a bond that is not a true peptide bond. Recently, an assay that combines the uses of a natural protein, the sensitivity of fluorescence, and the ease
Simple assay for proteases based on aggregation of stimulus-responsive polypeptides
Analytical chemistry, 2014
Unregulated changes in protease activity are linked to many diseases including cancer. Fast, accurate, and low-cost assays for detection of these changes are being explored for early diagnosis and monitoring of these diseases and can also be used as platforms for the discovery of new drugs. We report a new methodology for the simple detection and quantification of protease activity in buffer and human serum. The assay is based on recombinant diblock polypeptides that undergo temperature- or salt-triggered micellization in water. The coronae of the micelles are linked to the water-insoluble cores by a peptide substrate that is cleaved in the presence of the target protease. Protease cleavage of the diblock polypeptide triggers the aggregation of the core-forming segment, leading to a change in solution optical density, which can be used to detect the presence of, and to quantify the concentration of, protease. We used matrix metalloproteinase-1 (MMP-1) as a model protease and found p...