Anal. Chem. 2004, 76, 1429-1436
Fluorescence Quenching-Based Assays for Hydrolyzing Enzymes. Application of Time-Resolved Fluorometry in Assays for Caspase, Helicase, and Phosphatase Jarkko Karvinen,* Ville Laitala, Maija-Liisa Ma 1 kinen, Outi Mulari, Johanna Tamminen, Jorma Hermonen, Pertti Hurskainen, and Ilkka Hemmila 1
PerkinElmer Life and Analytical Sciences, Wallac Oy, P.O. Box 10, FIN-20101 Turku, Finland
We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energytransfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z′ factors. Time-resolved fluorometry is a technique based on the exceptionally long fluorescence lifetime characteristics of certain fluorophores.1 Some lanthanide ions, such as Eu3+, Tb3+, Sm3+, and Dy3+, are known for long lifetime fluorescence and have found wide use in diagnostics, research, and high-throughput screening (HTS).1,2 Background fluorescence originating for example from sample material, library compounds, and measurement cuvette has a short lifetime in nanosecond scale. The long lifetimes of lanthanide chelates enable a long delay, usually between 50 and 400 µs, before opening of the measurement window. Thus, time resolution reduces the background significantly compared to traditional prompt fluorescence measurements, enabling development of new applications including measurement of very low enzyme activities. Homogeneous time-resolved fluorescence energy* Corresponding author: (tel.) +358 2 267 8690; (fax) +358 2 2678 357; (email)
[email protected]. (1) Hemmila¨, I.; Mukkala, V-M. Crit. Rev. Clin. Lab. Sci. 2001, 38, 441-519. (2) Hemmila¨, I. Applications of Fluorescence in Immunoassays; Wiley-Interscience: New York, 1991. 10.1021/ac030234b CCC: $27.50 Published on Web 02/03/2004
© 2004 American Chemical Society
transfer (TR-FRET) assays utilizing nonradiative energy transfer between fluorescent lanthanide chelates and different acceptors are particularly suitable for measuring binding reactions and handling the large number of samples in HTS.3,4 However, for assays measuring enzyme-catalyzed hydrolysis or dissociation reactions, TR-FRET is not as well suited. Those assays require an approach where even a minor fraction of a hydrolyzed substrate can be distinguished from the bulk of unhydrolyzed substrate. The long decay behavior of lanthanide chelates results in particularly efficient energy-transfersor quenchingsreactions, as shown by enzymatic assays using FRET pair-labeled peptide substrate.5,6 We have also shown that TR-FQA (TruPoint) technology can be applied to multiplexing protease assays.7 To further study the quenching reaction, its utility to substrates of various sizes, and different enzymatic reactions, we chose three different assay models, caspase as an example of protease, helicase as an example of nucleic acid-modifying enzyme, and phosphatase as an example of a small substituent transfer reaction. Each of the enzymatic groups constitute important targets for drug discovery8-12 and also set different demands for the energy-transfer reactions due to energy-transfer (ET) distance, flexibility, or rigidity of the substrate structure and the control of labeling sites as defined or random. In the present study, we used various fluorescent and stable europium chelates as energy donors. Spectrally different fluorescent and nonfluorescent chromophores were tested as acceptors. The goal in the study was to develop assay systems for hydrolyzing enzymes that would (1) be sensitive enough, (2) produce an increasing response, (3) have a wide dynamic range obtained by (3) Mathis, G. Clin. Chem. 1993, 39, 1953-1959. (4) Hemmila¨, I.; Webb, S. Drug Discovery Today 1997, 2 (9), 373-381. (5) Karvinen, J.; Hurskainen, P.; Gopalakrishnan, S.; Burns, D.; Warrior, U.; Hemmila¨, I. J. Biomol. Screening 2002, 7 (3), 223-231. (6) Gopalakrishnan, S. M.; Karvinen, J.; Kofron, J. L.; Burns, D. J.; Warrior, U. J. Biomol. Screening 2002, 7 (4), 317-323. (7) Karvinen, J.; Elomaa, A.; Ma¨kinen, M.-L.; Hakala, H.; Mukkala, V.-M.; Peuralahti, J.; Hurskainen, P.; Hovinen, J.; Hemmila¨, I. Caspase Multiplexing: Simultaneous Homogeneous Time-Resolved Quenching Assay (TruPoint) for Caspases 1, 3 and 6. Anal. Biochem., in press. (8) Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43 (3), 305341. (9) Yao, N.; Weber, P.C. Antiviral Ther. 1998, 3 (Suppl 3), 93-97. (10) Kadare´, G.; Haenni, A.-L. J. Virol. 1997, 71 (4), 2583-2590. (11) Huisduijnen, R.; Bombrun, A.; Swinne, D. DDT 2002, 7 (19), 1013-1020. (12) Zhang, Z.-Y. Curr. Opin. Chem. Biol. 2001, 5, 416-423.
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substrates with high percentage of quenching, (4) be robust for the application used, and (5) and have potential for multiplexing using more than one label simultaneously. MATERIALS AND METHODS General Methods. All assays were measured using different versions of the VICTOR multilabel counter (PerkinElmer Life and Analytical Sciences, Wallac, Turku, Finland) and were performed in white or black 384-well plates (Wallac and OptiPlates, PerkinElmer Life and Analytical Sciences, Boston, MA) (see figure legends and below for details). All measurements were done with standard VICTOR excitation (340 nm) and emission (615 nm) Eu filters. Signal-to-background ratios were calculated from assays performed with and without enzyme. Chelates. All assays were developed using commercially available fluorescent and stable Eu chelates. The chelates are composed of polycarboxylate complexes containing an aromatic light-harvesting group (pyridine, terpyridine, or 1,3-dipyridylpyrazole) and are negatively charged and hydrophilic. All the chelates used (Eu-W1284, Eu-W8184, Eu-W14014) were obtained from PerkinElmer Life and Analytical Sciences, Wallac, and used as recommended by the manufacturer. Simplified structures of the chelates have been published earlier.7 Quenchers. In the study of short-distance energy transfer, different fluorescent and nonfluorescent chromophoric structures were tested. The chromophoric compounds used were dabcyl (succinimidyl ester and peptide building block, -dabcyl-R-FMOCL-lysine), isothiocyanate (ITC) activated tetramethylrhodamine (TMR), Alexa Fluor 633, 546, and 647, QSY 7, QSY 21 (Molecular Probes, Eugene, OR), and Cy5 (Amersham Biosciences, Uppsala, Sweden), all as succinimidyl esters. Spectra. Emission spectra for a typical Eu chelate was measured in 50 mM TSA buffer (50 mM Tris-HCl, 0.9% NaCl, 0.05% sodium azide, pH 7.75) using an LS-55 spectrofluorometer (PerkinElmer Life and Analytical Sciences, Shelton, CT). The Eu concentration was 980 nM. Absorption spectra for Cy5 was measured using Cy5 monofunctional reactive dye in PBS buffer (pH 7.0) with a Shimadzu UV2501 PC spectrophotometer. The concentration of the dye was 20 µg/mL. All other spectral data were from the Molecular Probes web site (www.probes.com) and are used with permission from Molecular Probes. Caspase Assay. The caspase-3 substrate (Ac-CDEVDK-NH2) was from Sigma-Genosys. Iodoacetamido (IAA) activated chelate Eu-W1284 was attached to cysteine of the substrate according to the manufacturer’s instructions. The product was purified with reversed-phase HPLC and confirmed with a MALDI-TOF mass spectrum (Centre for Biotechnology, Turku, Finland) (observed 1422.94, calculated 1422.22). The -amine of the lysine residue was further conjugated with different quenchers, QSY 7, tetramethylrhodamine, Alexa Fluor 546, and Cy5. All of these labelings were performed as recommended by the manufacturers using an approximately 3:1 molar ratio of dye compared to Eu-labeled peptide, purified with reversed-phase HPLC, and concentrated using a Hetovac vacuum drier (Heto-Holten, Denmark). Any ambiguous peaks from the HPLC were tested with the enzyme reaction. A dabcyl-quenched substrate, synthesized as described earlier,5 was also tested. The concentrations and quenching efficiencies of all the substrates were determined as described earlier.5 1430 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004
Figure 1. Principles of the protease, helicase, and phosphatase assays. (A) Caspase-3 assay. The assay is based on the recovery of the Eu fluorescence after separation of the Eu chelate and quencher due to enzymatic activity. (B) Helicase assay. Similar to caspase-3 assay, the Eu fluorescence recovers after separation of quencher and Eu strands by helicase. (C) Phosphatase assay. Assay principle relies on Eu-labeled substrate and quencher-labeled antiphosphotyrosine antibody. After removal of phosphate group by phosphatase, the antibody binding is diminished and the Eu fluorescence is not quenched.
The principle of the enzymatic assay is outlined in Figure 1A and has also been described earlier.5 Assays were performed on 384-well plates with a total volume of 20 µL and measured with a VICTOR2 multilabel counter. Caspase-3 concentration used in the assays varied between 0.25 and 10 ng/µL. The substrates were tested at concentrations ranging from 0.02 to 2000 nM (see figure legends for assay details). Helicase Assay. Oligonucleotides were synthesized using an Expedite 8909 oligonucleotide synthesizer (Applied Biosystems).
The synthesis of a 26-mer quencher strand was started with 3′PT-Amino-Modifier C6 CPG (Glenn Research) to enable the addition of the quenchers. The amino-modified oligonucleotide was purified with PAGE, concentrated under vacuum, and labeled with succinimidyl ester activated QSY 7, dabcyl, or TMR as recommended by the manufacturer to form the quencher strands. After purification with HPLC, the concentration of quencher strands was determined spectrophotometrically as advised by the dye manufacturer. Chelate W8184 was incorporated to the 5′ end of the detection strand (46-mer) as a building block during the synthesis as described earlier.13 After synthesis, europium was incorporated into the chelate and the product was purified with PAGE. The capture strand was from a commercial helicase kit (PerkinElmer Life and Analytical Sciences, Wallac). The helicase substrate was formed by allowing the complimentary Eu and quencher strands to anneal in a molar ratio of 1:3 at 35 °C for 3 h in 50 mM Tris-HCl, 0.5 M NaCl, pH 8. The quenching efficiency of each dye was determined by comparing the Eu fluorescence of the quencher strand annealed substrate to the fluorescence of the free Eu strand. The enzyme assays (Figure 1B) were performed using SV 40 large T antigen (CHIMERx). The buffer used was 20 mM Tris pH 7.5, 50 mM NaCl, 3 mM MgCl2, 0.5 mM ATP, 1 mM DTT, 0.1% Triton X-100, 10% glycerol, recommended in the PerkinElmer Life and Analytical Sciences helicase kit. Substrates with different quenchers were tested with 8 nM substrate and 80 nM enzyme concentration. The reaction was incubated at 35 °C and measured with a VICTOR2 counter. To prevent rehybridization of the strands separated by the enzyme, a QSY 7-labeled quencher strand was also tested with different capture strand concentrations. The enzyme amount was titrated by using 20-200 nM enzyme together with 8 nM substrate and 30 nM capture strand. Phosphatase Assay. A substrate for phosphatase CD-45 (H2NTSTEPQY(PO3H2)QPGENL-COOH), based on the negative regulatory site of pp60c-src 14,15 was synthesized with Applied Biosystems 433A peptide synthesizer using standard FMOC chemistry. Building blocks were from Novabiochem and PAL Support from Perseptive Biosystems. The product was characterized with an analytical reversed-phase HPLC and Applied Biosystems Mariner System 5272 mass spectrometer and used crude in labeling reaction. A control peptide with no phosphotyrosine was synthesized in a similar manner. The free amino terminus of the synthesized peptide was labeled with ITC-activated chelate Eu-W14014 as recommended by the manufacturer, purified with reversed-phase HPLC, and characterized and concentrated as described for caspase-3 substrate above. Anti-phosphotyrosine antibody PT-66 (Sigma, St. Louis, MO) was labeled with chromofores QSY 7, QSY 21, Alexa Fluor 633, Alexa Fluor 647, and Cy5 according to the manufacturers’ instructions. Either NAP-5 and PD-10 columns (Amersham Biosciences) or the gel filtration columns delivered with the labeling kits were used in the purification of the labeling reactions. The concentrations and degrees of labeling were determined spectrophotometrically as advised by the dye manufacturers. (13) Hovinen, J.; Hakala, H. Org. Lett. 2001, 3 (16), 2473-2476. (14) Roussel, R. R.; Brodeur, S. T.; Shalloway, D.; Laudano, A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10696-10700. (15) Ng, D. H. W.; Harder, K. W.; Clark-Lewis, I.; Jirik, F.; Johnson, P. J. Immunol. Chem. 1994, 179, 177-185.
Enzymatic assays were performed on black 384-well plates (PerkinElmer Life and Analytical Sciences, Wallac) with a total volume of 20 or 22.5 µL for quenching efficiency and enzyme assays, respectively. The measurements were carried out with a VICTOR2 V multilabel counter using two different delay times, 400 and 800 µs, and a counting window of 400 µs. The ability of the randomly labeled antibodies to quench Eu fluorescence (W14014) was first tested with 100 nM Eu-labeled phosphopeptide together with 100, 500, and 1000 nM labeled antibody. The buffer used was TSA + 0.1% Tween 20. The suitability of different quenchers for the phosphatase assay was also tested by spiking 10 nM nonphosphorylated substrate into the assay. After these initial testings, Cy5-labeled antibody was chosen to be used in the enzyme assay with phosphatase CD-45 (BIOMOL Research Laboratories). The principle of the assay is shown in Figure 1C. The assays were performed in a buffer recommended by the enzyme supplier (100 mM Hepes, pH 7.0, 1 mM EDTA, 5 mM DTT, 0.05% Tween 20), unless otherwise stated (see figure legends), using 50 and 100 nM Eu-labeled phosphopeptide and 500 ng/mL enzyme. After 1-h incubation at 30 °C, the quencher antibody was added into the reaction (final concentrations 500 and 1000 nM). This mixture was incubated for 1 h before measurement with VICTOR2 V. The assay was further optimized by enzyme titration using 100 nM substrate and 1000 nM quencher-antibody concentration. Otherwise, the assay was performed as above. To confirm that the observed increase in signal was due to the enzyme activity, an inhibition curve with known inhibitor RWJ6047516 (BIOMOL Research Laboratories) was measured with 50 nM substrate, 1000 nM Cy5 quencher antibody, and 0.4 ng of enzyme. RESULTS AND DISCUSSION Spectra. Spectra of a representative Eu chelate and different fluorophores are presented in Figure 2. There are only minimal changes in the location of the Eu emission peaks between different chelates17 even though there might be differences between the relative sizes of the peaks. All of the assays described here have been measured using the 5D0 f 7F2 transition emitting at 615 nm. The tested quenchers dabcyl, TMR, and Alexa Fluor 546 show minimal overlapping with the emission spectra of the europium. QSY 7 has partial and Alexa Fluor 633, Alexa Fluor 647, QSY 21, and Cy5 significant overlapping with the europium emission. All of the quenchers tested were capable of quenching the Eu fluorescence with one or more of the model assays (Tables 1-3 and below). Caspase Assay. The synthesis of the caspase-3 substrate was started by adding an IAA-activated Eu chelate W1284 into the cysteine residue of a commercial peptide. The quenchers QSY 7, TMR, Alexa Fluor 546, and Cy5 were then incorporated into this Eu-labeled peptide. In HPLC purification, all the chromophores, except TMR, produced one colored peak with significant Eufluorescence. Labeling with rhodamine produced two peaks probably relating to two different rhodamine isomers. Both of them were tested with the enzyme reaction. (16) Beers, S. A.; Malloy, E. A.; Wu, W.; Wachter, M. P.; Gunnia, A.; Cavender, D.; Harris, C.; Davis, J.; Brosius, R.; Pellegrino-Gensey, J. L.; Siekierka, J. Bioorg. Med. Chem. 1997, 5 (12), 2201-2211. (17) Freeman, J. J.; Crosby, G. A. J. Phys. Chem. 1963, 67, 2717-23.
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Figure 2. Emission spectra of a representative Eu chelate W8044 and absorption spectra of quenchers tested. The spectra of the chelate and Cy5 were measured as explained in Materials and Methods, Data for other fluorophores are from the Molecular Probes web site (www.probes.com) and used with permission.
Figure 3. (A) Comparison of different quenchers tested in the caspase-3 assay with different substrate concentrations. The assay was performed on WALLAC white 384-well plates with 500 pg/µL caspase-3 using two replicas. Presented data are after 30-min incubation at +37 °C and were measured with 400-µs window after 400-µs delay using VICTOR2 multilabel reader. QSY 7 was found to be the most efficient quencher of Eu fluorescence. (B) Rescale of (A) for QSY 7 with a logarithmic scale.
Table 1. Comparison of Different Quenchers with Caspase-3 Assay S/B with [substrate] quencher
quenching efficiency (%)
max S/B
200 pM
200 nM
dabcyl Cy5 QSY 7 TMR (fr2) TMR (fr1) Alexa Fluor 546
nd 99.2 99.97 99.4 95.3 98.9
114 72 696 42 9 104
6.4 7.5 13.8 4.1 1.5 11.5
92.5 58.2 560.3 41.5 7.2 101.3
The quenching efficiencies and signal-to-background ratios (S/ B) measured are presented in Table 1 and in Figure 3. QSY 7 proved superior to the other quenchers consistently producing S/B levels above 600 with quenching efficiencies above 99.9%. Moreover, it produced a very stable signal with ∼10% decrease in the S/B ratio over a period of 24 h (data not shown). A caspase-3 1432 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004
assay using the QSY 7-quenched substrate produced a linear response approaching 3 orders of magnitude, the sensitivity being better than 5 pg/well (250 fg/µL, 8.6 pM) caspase-3 (Figure 4). The statistical evaluation by the Z′ value18 with 12 replicates gave an excellent value of 0.975. Caspase substrate is a typical example of a relatively short and flexible substrate showing highly efficient quenching even with spectrally minimally overlapping quenchers. According to Fo¨rster’s law on energy transfer, there should be spectral overlapping between the donor and the acceptor. Another proposed quenching mechanism is based on ionic contact of the acceptor and the donor.19 From the quenchers tested dabcyl, Alexa 546, and TMR show only a minimal, QSY 7 partial and Cy5 significant overlapping with the emission spectra of the used Eu chelate (Figure 2). However, all of the quenchers were capable of accepting energy (18) Zhang, J.-H.; Chung, T. D. Y.; Oldenburg, K. R. J. Biomol. Screening 1999, 4, 67-73. (19) Yaron, A.; Carmel, A.; Katchalski-Katzir, E. Anal. Biochem. 1979, 95, 228235.
Figure 4. Enzyme titration of caspase-3. The assay was performed on black 384-well plate with 200 nM substrate concentration and differing amounts of caspase-3. Assay was measured with VICTOR2 using TR-FQA protocol after 30-min incubation at 37 °C.
Figure 5. Effect of capture strand on the S/B of the helicase assay. Assay was performed on a white Packard 384-well Optiplate with 8 nM QSY 7-quenched substrate and 80 nM enzyme concentrations, respectively. The plate was incubated for 2 h at 35 °C before measurement with VICTOR2 reader using 50-µs delay with 50-µs measurement window.
Table 2. Comparison of Different Quenchers with Helicase Assay quencher
quenching efficiency (%)
max S/B
QSY 7 TRITC dabcyl
98.88 94.43 99.28
59.8 19.6 85.4
from the chelate even though with different efficiencies. Dabcyl and QSY 7 have a positive, and Alexa Fluor 546 and Cy5 a negative charge. TMR is carrying a neutral total charge. The flexibility of the substrate probably enables ionic interaction between the positive charge of dabcyl or QSY 7 and the negatively charged chelate, which could account for the extraordinarily efficient quenching even without spectral overlapping. Based on our results with the caspase-3 assay, the mechanism of ionic interaction results in more efficient quenching compared to Fo¨rster’s energy transfer with the Eu chelate used and thus produces higher S/B ratio when using short peptide substrates. Helicase Assay. Helicase substrates were formed by annealing an Eu-W8184-labeled 44-mer strand with a quencher-labeled 26-mer strand. The quenching efficiencies determined by different quenchers are presented in Table 2 together with maximal S/B ratios. The quenching efficiencies of QSY 7 and dabcyl were relatively similar, whereas that of TMR was significantly lower. Similar differences were found in maximal S/B ratios (Table 2). Dabcyl was found to be the most efficient quencher, but since we have earlier found problems with the signal stability of dabcyllabeled caspase-3 substrate (J. Karvinen, unpublished results), we chose QSY 7 for further testing. The addition of the capture strand into the assay lowered the S/B ratio (Figure 5) but increased the signal stability of the assay (Figure 6A). The decrease in the S/B ratio was probably caused by the prevention of rehybridization after spontaneous separation of the quencher and Eu strands, which results in slightly increased background (Figure 6B) in assays with capture strand. Results from the enzyme titration are presented in Figure 7. The calculated Z′ value with 12 replicates was 0.92.
Helicase has a relatively rigid double-helix structure as a substrate and is an example of DNA-based enzymatic application. In this application, the donor-acceptor distance can be stipulated quite widely and optimized for optimal energy transfer and decay time. Even though the distance between the Eu chelate and quencher is probably much shorter in the helicase than in the caspase substrate, both the quenching efficiencies and S/B ratios were significantly lower. One possible explanation for this is the better light efficiency of Eu-W8184 compared to Eu-W1284,20,21 which decreases the quenching efficiency. Another possible reason is the strong negative charge of DNA, which may disturb the interaction between the Eu chelate and the quencher preventing partly the ionic quenching.19 The efficiency of annealing is not 100%, and this also results an increase in the background signal. The nearly 10-fold decrease in the S/B ratio compared to the caspase assay is most probably due to a combination of all these effects. The helicase model (DNA-DNA hybridization) can also be used as a tool to study both energy-transfer distances and the contribution of spectral overlapping. Such work is currently going on. Phosphatase Assay. The phosphatase assay was developed with a nine-dentate Eu-W14014 chelate used to label a phosphatase substrate peptide and quencher-labeled anti-phosphotyrosine antibodies. Table 3 illustrates the degrees of labeling, the quenching efficiencies of antiphosphotyrosine antibody PT66 labeled with different quenchers, and the S/B ratios with 10 nM spiked nonphosphorylated substrate. Only Cy5-, Alexa 633-, and Alexa 647-labeled antibodies showed significant ability to quench Eu fluorescence in this assay. The Cy5-labeled antibody was chosen to be tested with the enzyme assay for the following reasons. The emission spectrum of Alexa 633 partly overlaps with the europium emission and therefore causes some increase in (20) Takalo, H.; Mukkala, V.-M.; Mikola, H.; Liitti, P.; Hemmila¨, I. Bioconjugate Chem. 1994, 5, 278-282. (21) Mukkala, V.-M.; Helenius, M.; Hemmila¨, I.; Kankare, J.; Takalo, H. Helv. Chim. Acta 1993, 76, 1361-1378.
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Figure 6. Effect of capture strand on specific (A) and background (B) signals of helicase assay. The background signals were measured from wells without the enzyme. The substrate used was quenched with QSY 7 and assay protocol was as indicated in legend for Figure 5.
Table 3. Properties of Different Quenchers in the Phosphatase Assay quenching efficiencyb (%) quencher
100 nM
500 nM
1000 nM
labeling degree
S/Bc
Alexa Fluor 647 QSY 21 Alexa Fluor 633 CY-5 QSY 7a
38.7 33.6 48.0 65.4 14.2
89.1 46.5 86.5 93.2 43.2
97.3 68.9 91.7 98.5 59.2
5.6 2.5 2.9 7.5 3.5
3.9 1.7 3.6 6.1 nd
a Measured with 400/400 window; all others measured with 800/ 400 window. b 100 nM p-peptide, different concentrations of anti-pantibody labelled with quencher. c Measured with 10 nM non-p-peptide spiked into 100nM p-peptide.
Figure 7. Enzyme titration with helicase assay. Assay conditions were 8 nM QSY 7 substrate, 20-200 nM SV-40 large T antigen enzyme, 30 nM capture strand in helicase assay buffer (see Material and Methods). Assay was incubated in 35 °C and measured with VICTOR2 after 2 h using 50-µs delay and 50-µs measurement window. Plate used was white WALLAC 384-well plate.
the background, and Alexa 647 also emits some energy-transfer signal. Cy5 behaved like a “dark quencher” with no energy-transfer signal at 665 nm (data not shown). It has been reported that with higher labeling degrees the Cy5 molecules undergo self-quenching.22 When six Cy5 molecules were coupled to IgG, the resulting quenching efficiency was 97%, mainly due to an inner-filter effect.22 This self-quenching, however, did not affect the ability of Cy5 to quench Eu fluorescence in the phosphatase assay developed. In the enzymatic assay, the dephosphorylation of phosphopeptide by CD-45 was monitored with two different measurement windows and two different substrate and quencher-antibody concentrations (Figure 8A and B). The 1000 nM quencherantibody and 100 nM substrate concentrations produced the highest S/B ratio. There was also a significant difference between (22) Gruber, H. J.; Hahn, C. D.; Kada, G.; Riener, C. K.; Harms, G. S.; Ahrer, W.; Dax, T. G.; Knaus, H.-G. Bioconjugate Chem. 2000, 11, 696-704.
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the measurement windows. The measurement after a 800-µs delay resulted in ∼2 times better signal-to-background ratios (Figure 8B). The calculated Z′ value (12 replicates) was 0.91, indicating an excellent assay. The signal is dependent on the enzyme concentration as shown by a enzyme titration curve (Figure 9). Even as low an enzyme amount as 400 pg/well gave a reasonable signal. The inhibition curve of a known inhibitor RWJ-60475 is presented in Figure 10. The IC50 value calculated with GraphPad Prism (GraphPad Software Inc.) was 3.3 µM, which is consistent with the 2 µM presented in the literature.16 The inhibition curve proved that the produced signal decreases as a function of the inhibitor concentration, and the assay thus truly measures enzymatic activity. Phosphatase activity was measured similarly to caspase by using peptide substrates. An anti-phosphotyrosine antibody was utilized as a specific carrier of randomly distributed quenchers (Figure 1C). Due to random labeling of the antibody and of its size, there is a significantly longer ET distance. Due to the longer ET distance, the assay required more efficient energy transfer and larger spectral overlapping compared to protease and helicase assays. For example, QSY 7-labeled antibody was not able to quench the Eu-labeled phosphatase substrate at all even though it has partial overlapping with the Eu emission spectra and it was
Figure 8. Testing of phosphatase assay with CD45. The assay was performed on black WALLAC 384-well plate in total volume of 25 µL. Phosphatase substrate (50 and 100 nM) was incubated at 30 °C with 500 ng/mL CD45 phosphatase for 1 h. After addition of 5 µL of Cy5labeled quencher antibody (final concentrations 500 and 1000 nM), the plate was incubated for an other 2 h (RT) and measured with VICTOR2 V with 400/400 (A) and 800/400 (B) measurement windows.
Figure 9. Enzyme titration with phosphatase assay. Substrate concentration was 100 nM and plate used black WALLAC 384-well. Plate was incubated with enzyme and substrate for 20 min at 35 °C before addition of 1000 nM Cy5-labeled quencher antibody. After 1-h incubation with the antibody, the plate was read with VICTOR2 V using 800-µs delay and 400-µs measurement window.
found to be an extremely potent quencher for the protease and helicase assays. In addition to only partially overlapping spectra, also the labeling degree for QSY 7 was relatively low. All efforts to increase the number of quencher molecules per antibody lead to precipitation of the antibody due to the hydrophobicity of the dye. In addition to different demands for spectral properties of the quenchers, the longer ET distance in the phosphatase assay also slowed the quenching process. With the phosphatase assay developed, a delay up to 800 µs was needed for efficient quenching, reflecting the longer decay in energy-transfer pair. For comparison, with the caspase assay a quenching level of over 99% was achieved even after a 50-µs delay before the measurement window. Phosphatase assay required a significantly higher quencher concentration compared to caspase and helicase assays. This,
Figure 10. Inhibition of CD45 phosphatase by RWJ-60475. The assay was performed in 100 mM HEPES, pH 7.0, 1 mM EDTA, 2.5 mM DTT, and 0,02% nonionic detergent P-40 (Sigma). Enzyme (0.4 ng/µL) and inhibitor were incubated for 30 min before addition of substrate to final concentration of 100 nM. After 1-h incubation at 30 °C, Cy5-labeled quencher antibody (1000 nM) was added into the wells. Assay was measured after 1-h incubation using VICTOR2 V with 800-µs delay and 400-µs measurement window. IC50 value calculated with GraphPad Prism was 3.3 µM.
increased dynamic quenching resulted in lower signal levels with the phosphatase assay (data not shown). The dynamic quenching did not prevent the use of the assay developed as both the enzyme assays (Figures 8 and 9) and inhibitor titration curve (Figure 10) resulted with good assay windows. CONCLUSIONS Assays for monitoring protease, helicase, and phosphatase assays based on time-resolved fluorescence quenching were developed, and the efficacy of different dyes to quench Eu fluorescence was also tested. All of the assays developed are homogeneous and nonradioactive with excellent Z′ values and high signal-to-background ratios. They also produce an increasing signal, which usually is favorable for HTS data processing. All the assays developed had different demands for the optimal quencher depending on the ET distance and the ionic environment around the energy donor (Eu chelate) and acceptor (quencher). Analytical Chemistry, Vol. 76, No. 5, March 1, 2004
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Assays having long ET distance, for example, the phosphatase assay, seem to demand that the quenchers have overlapping spectra with the emission of the chelate for efficient quenching and are based on traditional Fo¨rster’s quenching. On the other hand, when the ET distance is short, the quenching based on ionic interaction is more efficient, as proved in the caspase assay. The optimal quencher for Eu chelates used would probably have at least partial overlapping with the emission spectra of the europium and a positive charge to enable also ionic interaction. From the quenchers tested, QSY 7 fulfilled these requirements
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best and also proved to be the most efficient quencher in the protease assay. ACKNOWLEDGMENT We thank K. Nenonen, P. Pulli, and R. Heilimo¨ for excellent technical assistance, K. Loman for synthesizing the phosphatase substrates, and Dr. H. Hakala for mass spectrometry analysis. Received for review June 17, 2003. Accepted November 18, 2003. AC030234B
