Fluorescence Quenching-Based Assays for Hydrolyzing Enzymes. Application of Time-Resolved Fluorometry in Assays for Caspase, Helicase, and Phosphatase (original) (raw)
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Analytical Biochemistry, 2007
Fluorescence change is convenient for monitoring enzyme kinetics. Unfortunately, it looses linearity as the absorbance of the fluorescent substrate increases with concentration. When the sum of absorbance at excitation and emission wavelengths exceeds 0.08, this inner filtering effect (IFE) alters apparent initial velocities, K m , and k cat . The IFE distortion of apparent initial velocities can be corrected without doing fluorophore dilution assays. Using the substrate's extinction coefficients at excitation and emission wavelengths, the inner filter effect can be modeled during curve fitting for more accurate Michaelis-Menten parameters. A faster and simpler approach is to derive k cat and K m from progress curves. Strategies to obtain reliable and reproducible estimates of k cat and K m from only two or three progress curves are illustrated using matrix metalloproteinase-12 and alkaline phosphatase. Accurate estimates of concentration of enzyme active sites and specificity constant k cat /K m (from one progress curve with [S] ≪ K m ) confer accuracy, freedom of choices of [S], and robustness to k cat and K m globally fitted to a few progress curves. The economies of the progress curve approach make accurate k cat and K m more accessible from fluorescence measurements.
Analytica Chimica Acta, 2007
The p53 protein is a tumor suppressor that protects the organism against malignant consequences of DNA damage. Interaction of p53 with numerous cellular or viral proteins regulates its functional activity either positively or negatively. An approach leading to identification of such protein interactions directly in a cell extract could be of help in the development of screening assays to search for drugs acting on p53 in its cellular environment, either by disrupting its association with inhibitory proteins or by increasing its affinity for activating proteins. We show that the homogeneous time-resolved fluorescence (HTRF) assay based on the time-resolved amplified cryptate emission (TRACE) technology allows identification of such an interaction by simply adding a mixture of two labeled monoclonal antibodies, directly in a cellular extract. We validate this assay by studying p53/SV40-LTAg interactions. The antibodies directed against genuine p53 and SV40-LTAg epitopes were labeled with europium cryptate (donor) and XL665, a crosslinked allophycocyanin (acceptor), respectively. We demonstrated that a nonradiative energy transfer occurs between labeled antibodies only when p53 interacts with SV40-LTag, which opens up the possibility of extending this approach to other p53 partners to search for drugs that restore p53 tumor-suppressor activity. Ó
Fluorescence tools to measure helicase activity in real time
Methods, 2010
Methods are described to show how different fluorescent labeling strategies can be used to probe various aspects of the helicase mechanism. Fluorophores on the adenine nucleotide, the DNA or the helicase can modify the activity of the system to a greater or lesser extent. Reagentless biosensors, binding proteins that are labeled with a fluorophore, target products of the helicase reaction, namely ADP, inorganic phosphate or single-stranded DNA, and can be used to measure rates of product formation with little interference to the system. Protocols are described to examine ATP usage and translocation speeds and also to investigate details of the ATP hydrolysis cycle. The methods are described in terms of PcrA, a bacterial DNA helicase that moves in single base steps along either single-stranded or double-stranded DNA, hydrolyzing one ATP per base moved.
Analytical Biochemistry, 2012
There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce on separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.
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
Biochemical Journal, 2001
Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20-60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as Abbreviations used : CCD, charge-coupled device ; CRET, chemiluminescence resonance energy transfer ; EGFP, enhanced green fluorescent protein (GFP human codon usage, with the mutation S65T) ; EGCAq, EGFP-caspase-3 linker-aequorin ; EGTAq, EGFP-α-thrombin linker-aequorin ; FMK, fluoromethylketone ; FRET, fluorescence resonance energy transfer ; GFP, green fluorescent protein ; GAq, GFP-aequorin ; GCAq, GFP-caspase-3 linker-aequorin ; GTAq, GFP-α-thrombin linker-aequorin ; LAq, firefly-luciferase-aequorin ; Z-, benzyloxycarbonyl. ).
Analytical Biochemistry, 2007
Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as k cat /K m values, which ranged from 0.2 to 80 · 10 6 M À1 min À1 . The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.
Molecular Pharmaceutics, 2009
We have developed a generic approach to determine enzyme activities in vitro and monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)-acceptor (Me 2 NCp) near infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me 2 NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me 2 NCp)-DEVD-APK(CbOH)-NH 2 (5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5 was cleaved more readily than 4, yielding k cat and K M , values of 1.02 ± 0.06 s −1 and 15 ± 3 μM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in >2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitro kinetics measurements to in cellulo and in vivo localization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitro and monitor treatment response in living organisms.