- Email: [email protected]
In vitro assays for the determination of histone deacetylase activity
Methods 36 (2005) 332–337 www.elsevier.com/locate/ymeth
In vitro assays for the determination of histone deacetylase activity Birgit Heltweg a, Johannes Trapp b, Manfred Jung b,¤ a
b
Fred Hutchinson Cancer Research Center, Seattle, USA Department of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany Accepted 30 March 2005
Abstract Histone deacetylases are important regulators of transcription and an emerging target for anticancer drugs. We present an overview over various assay formats that include radiolabelled histones, oligopeptides, and small molecules as substrates. The advantages and disadvantages of the various formats in terms of, e.g., substrate availability, throughput or subtype selectivity are discussed. Detailed procedures for various assay types that can be used for diVerent problems, such as library screening or Xuorescent inhibitor testing, are given. We present a new protocol for a simple high-throughput assay for NAD+-dependent (class III) histone deacetylases, also termed sirtuins. 2005 Elsevier Inc. All rights reserved. Keywords: Histone deacetylase; Sirtuin; Assay; Screening; Aminomethylcoumarin
1. Introduction Histone deacetylases (HDACs) are enzymes that contribute to the posttranslational modiWcations of histones and various non-histone proteins. Deacetylation leads to altered states of conformation and/or activity for the proteins in question [1]. HDACs have been recognized mostly as transcriptional repressors and the aberrant recruitment of deacetylase activity has been linked to distinct leukemia types as a basis for the diseases on a molecular level. Inhibitors of histone deacetylase generally lead to a relief of transcriptional repression which in turn leads to apoptosis and/or diVerentiation in cancer cells. First clinical studies on HDAC inhibitors as new anticancer agents are therefore under way [2]. Eleven subtypes in two classes of those deacetylases that are zinc-dependent amidohydrolases have been discovered so far. There is also a third class of histone deacetylases, also called sirtuins, that requires NAD+ for its catalysis
*
Corresponding author. Fax: +49 761 203 6321. E-mail address: [email protected] (M. Jung).
1046-2023/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2005.03.003
which is not sensitive to class I and II inhibitors [3]. Considering the increasing interest in those enzymes it is very important to have eYcient assays for the detection of their activity at hand. This is needed for both protein isolation and characterization as well as drug screening. We present a comprehensive survey of the existing assays and a new method for the homogeneous measurement of sirtuin activity.
2. Radioactive substrates Traditional assays for the in vitro measurement of histone deacetylase (HDAC) activity involve radioactively labeled histones isolated from chicken reticulocytes or cell culture as the substrates [4]. The substrate properties are diYcult to standardize and show a high batch-to-batch variation. The production of the labeled chicken histones involves pretreatment of the chickens with phenylhydrazine which leads to severe anemia. The animals have to be killed during collection of the cells. The preparation of recombinant histones has recently overcome some of those problems [5]. As an alternative
B. Heltweg et al. / Methods 36 (2005) 332–337
to the histones chemically [3H]-acetylated oligopeptides with 8 [6] or 24 [7] amino acid residues that are derived from histone amino acid sequences have been introduced. They still bear the disadvantages of the use of radioactivity in terms of exposition of personnel and waste management. Additionally, the preparation of those peptides requires sophisticated laboratory equipment or they have to be purchased for a very high price. The deacetylase activity is monitored with both histones and peptides by the extraction of tritiated acetic acid from acidic medium and scintillation counting. This procedure is very time-consuming and therefore not suitable for a high sample throughput. There is a high-throughput version with a biotinylated and tritiated acetyl-histone peptide fragment available that makes use of a scintillation proximity assay [8]. This allows for a large number of samples but the other disadvantages of radioactive oligopeptides are retained.
3. Non-isotopic substrates Oligopeptides that are not radioactively labeled have also been used as in vitro HDAC substrates, e.g., Xuorescein-labeled octapeptides that were monitored by HPLC and Xuorescence detection [9]. Generally, these peptides are rather poor substrates and those methods also have only a low-throughput. They oVer, however, the potential of investigating lysine selectivity for various deacetylases or acetyltransferases. Tripeptides that contain a C-terminal acetyl-lysinecoumarinylamide have been described in a new homogeneous assay [10]. The detection reagent contains trypsin that is able to recognize the oligopeptide as a substrate only if the lysine has been deacetylated. Upon cleavage by trypsin the aminocoumarin is released and can be determined in a homogeneous fashion. A more detailed description concerning its application in high-throughput screening has been reported as well [11]. A similar kit is commercially available for both class I and II, respectively, II enzymes (CycLex). Further Xuorescence and absorption based kits are available by Biomol that work similarily, in part using larger peptidic substrates. A dually labelled sirtuin peptide substrate whose Xuorescent label is internally quenched is cleaved by an endopeptidase to yield a Xuorescent fragment upon deacetylation [12]. Recently, an acetylated lysine derivative, Boc(Ac)LysAMC, also termed MAL, had been introduced as the Wrst non-isotopic substrate for histone deacetylases [13,14]. It is easily accessible by a one-step synthetic procedure and is also commercially available. As the deacetylated metabolite shows the same Xuorescence properties as the parent substrate the conversion was initially monitored by an extraction/HPLC protocol. The use of an internal standard led to increased precision for the extraction proce-
333
dure [15]. The procedure was optimized by a new extraction protocol that allowed for a plate reader measurement[16] instead of a HPLC/Xuorescence detection quantiWcation. The original internal standard for that protocol BODIPY 530/550 had to be substituted by Eosin Y [17] as the former is not commercially available anymore. Alternatively, a derivatization reaction can be applied to achieve a homogeneous assay, the so-called HDASH-procedure [18]. In that version, the Xuorescence of the resulting metabolite is quenched by addition of naphthalene dicarboxaldehyde (NDA), an amine detection reagent, and the remaining substrate can be quantitated without separation. MAL can also be determined using the trypsin assay. It is a better substrate for HDAC than the tripeptides but its metabolite is not a very good substrate for trypsin [10]. The diversity of detection protocols allows for an optimized assay depending on the nature of the problem. The HPLC protocols can be applied when the simultaneous detection of substrate and metabolite is desired, when highly Xuorescent compounds should be checked for inhibition or when a plate reader is not available. The extraction/plate reader protocol is used especially when only small amounts of enzyme are available. It is valuable when diVerent samples with diVerent protein contents are screened, e.g., during chromatographic enzyme puriWcation. The homogeneous protocol is best used in inhibitor screening when a high-throughput is desired. Recently, we have introduced Z-(Ac)Lys-AMC, also termed ZMAL [19], as an optimized analogue of MAL. ZMAL is converted by human histone deacetylases faster and to a greater extent, especially valuable is the fact that it is in contrast to MAL a good substrate for the NAD+-dependent class III histone deacetylases (also called sirtuins) that have a completely diVerent mechanism of catalysis [3]. While ZMAL is deacetylated equally well by HDAC1 as a class I deacetylase and by HDAC6 as a class IIa enzyme a moderate subtype selectivity towards HDAC6 was observed for MAL. Similar substrates with a much more pronounced subtype selectivity have been published recently[20,21]. The HPLC and the homogeneous assay have been tested with equimolar amounts of ZMAL instead of MAL and give similar results. The extraction/ plate reader method is expected to behave similarly as well. The NDA detection method does not work with sirtuins. But the deacetylated metabolite of ZMAL, ZML, is a very good substrate for trypsin, and thus, a homogeneous sirtuin assay can be performed with the simple and inexpensive substrate ZMAL (see Table 1).
4. HPLC method without extraction 4.1. Materials, buVers, and enzymes Fluorescent histone deacetylase substrate MAL (Calbiochem): 12.6 mM in DMSO or ethanol, stored at
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Table 1 ClassiWcation of HDAC assays (for details see the corresponding paragraphs of the manuscript) Substrates
Preparation
Commercial availability
Assay and detection
Throughput
Remarks
Radiolabelled histones
From chicken blood, cell culture or recombinant; labelling with [3H]acetyl-CoA and histone acetyltransferase
Components
Extraction and scintillation counting
Low
Native substrate radioactive waste
Histone peptides Unlabelled Radiolabelled
Radiolabelled and biotinylated Fluorescence labelled
Often low reactivity Peptide synthesis Peptide synthesis, [3H]acetyl-CoA and HAT activity or [3H]acetate and chemical labelling Peptide synthesis, biotinylation
Yes Components
HPLC, UV detection Extraction and scintillation counting
Low Low
Components
Scintillation proximity HPLC and UVdetection Peptidase digestion and plate reader Xuorescence
High
Peptide synthesis, labelling Some as a kit
Small molecule Xuorescent substrates Tripeptide-AMCderivatives Lysine-AMCderivatives
Radioactive waste
Low High
Sirtuin substrates available Usually high reactivity
Oligopeptide synthesis
Some as a kit
One to two step syntheses
Components, some as kit
¡20 °C. Internal standard 7-hydroxycoumarin (Janssen, Germany): 6.3 mg/mL in DMSO, stored at ¡20 °C. Histone deacetylase, if not otherwise obtained, from rat liver (see literature [16] for protocol, or commercially available from Calbiochem, USA, or Alexis, Switzerland). Incubation buVer: 1.4 mM NaH2PO4, 18.6 mM Na2HPO4, pH 7.9, 0.25 mM EDTA, 10 mM NaCl, 10% (v/v) glycerol, and 10 mM mercaptoethanol. Stop mixture: 1 M HCl/0.4 M sodium acetate. HPLC system with a Shimadzu RF 535 Xuorescence detector (excitation wavelength 330 nm, emission wavelength 395 nm) and a Multospher RP-18, 5 m column (250 £ 4 mm, CS-Chromatographie, Germany) with a guard column of the same material (5 £ 4 mm). Chromatography eluent: acetonitrile/water/triXuoroacetic acid (55/45/0.01 v/v). Stock solutions of inhibitors in DMSO or methanol/ethanol.
2.
3.
4.
4.2. Procedure 5. 1. Take an aliquot of 12 L of the MAL stock solution and an al i quot of 15 L of the stock so lu tion of 7-hydroxycoumarin and add incubation buVer to a total volume of 1 mL. For inhibitor screening, dilute
6. 7.
plate reader Xuorescence HPLC and Xuorescence detection or extraction and plate reader Xuorescence
High
Tryptic or chemical detection, plate reader Xuorescence
High
Lowmedium
Sirtuin substrates and HDAC class I and II selective substrates available, components much cheaper than kits
the inhibitors with the incubation buVer to a concentration which is 12-fold higher than the desired assay concentration (dilute at least 1:10, as ethanol and DMSO from the stock solution of the inhibitor interfere with the enzyme). For standards without enzyme activity, take 110 L incubation buVer, add 10 L of the substrate/internal standard solution and mix well. Chill on ice. For inhibitor samples, take 100 L of enzyme or dilute the enzyme to 100 L with incubation buVer for each sample. Add 10 L diluted inhibitors and mix gently. Keep on ice for 10 min. Add 10 L of the substrate/internal standard solution and mix gently. Allow to stand at 4 °C for another 15 min. As enzyme activity standards, take 100 L of enzyme or diluted enzyme and add 10 L of the buVer. Keep on ice for 10 min. Add 10 L of the substrate/internal standard solution and mix gently. Allow to stand at 4 °C for another 15 min. Incubate the samples and standards for the desired length of time at 37 °C, usually 90 min. Stop the reaction by addition of 1000 L of acetonitrile and vortex approximately 30 s. Centrifuge at 10,000 rpm for 5 min.
B. Heltweg et al. / Methods 36 (2005) 332–337
8. Take 600 L of the supernatant and inject 20 L onto the HPLC system (Xow rate 0.5 mL/min). Retention time is 5.06 min for the deacetylated substrate, 5.64 min for the internal standard, and 7.09 min for the substrate.
5. HPLC extraction method 5.1. Materials, buVers, and enzymes Stop mixture: 1 M HCl/0.4 M sodium acetate. HPLC system: Shimadzu RF 535 Xuorescence detector (330 nm excitation wavelength, 395 nm emission wavelength), Lichrosorb RP-18, 5 m column (150 £ 3 mm, Knauer, Germany) with a guard column of the same material (5 £ 3 mm). Chromatography eluent: acetonitrile/water (40/60 v/v). 5.2. Procedure 1. Prepare samples and standards and incubate at 37 °C as described in HPLC method without extraction. 2. Stop the reaction by addition of 72 L of stop mixture and mix well. 3. Add 800 L ethyl acetate and vortex approximately 30 s. 4. Centrifuge at 10,000 rpm for 5 min. 5. Take 200 L of the upper phase and remove ethyl acetate by a stream of nitrogen. 6. Dissolve residue in 600 L of the chromatography eluent, mix well, and inject 20 L onto the HPLC system (Xow rate 0.6 mL/min). Retention time of the internal standard is 2.17 and 3.47 min for the substrate.
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2. For standards without enzyme activity, take 110 L of incubation buVer, add 10 L of the substrate/internal standard solution, and mix well. Chill on ice. 3. For inhibitor samples, take 100 L of enzyme or dilute the enzyme to a total volume of 100 L with incubation buVer for each sample. Add 10 L of diluted inhibitors and mix gently. Keep on ice for 10 min. Add 10 L of the substrate/internal standard solution and mix gently. Keep on ice for another 15 min. 4. For enzyme activity standards, take 100 L of enzyme or diluted enzyme and add 10 L of incubation buVer. Keep on ice for 10 min. Add 10 L of the substrate/internal standard solution and mix gently. Keep on ice for another 15 min. 5. Incubate samples and standards for desired length of time at 37 °C, usually 90 min. 6. Stop the reaction by addition of 400 L of 1 M HCl and mix well. 7. Add 800 L ethyl acetate and vortex approximately 30 s. 8. Centrifuge at 10,000 rpm for 5 min. 9. Take 400 L of upper phase and evaporate ethyl acetate by a stream of nitrogen. 10. Dissolve residue in 600 L acetonitrile/buVer mixture, vortex, and transfer 250 L into microplate. Read the plate at 330/390 nm.
7. Homogeneous assay for class I and II deacetylases (HDASH) 7.1. Materials, buVers, and enzymes
Internal standard Eosin Y (Merck, Germany): 9.3 mg/mL in ethanol. Microplate reader Polarstar Galaxy (BMG Labtechnologies, Germany) with an excitation Wlter of 330 nm and an emission Wlter of 390 nm. Black 96-well microplates (BMG Labtechnologies, Germany). Solvent: 1 part of buVer (pH 8, 0.5 mM KH2PO4, 0.46 mM NaOH) is diluted with 59.4 parts of water and this is then mixed with 39.6 parts of acetonitrile.
Histone deacetylase in phosphate buVer or another buVer (e.g., Hepes) without primary amines (do not use tris buVer!). Incubation buVer: 1.4 mM NaH2PO4, 18.6 mM Na2HPO4, pH 7.9, 0.25 mM EDTA, 10 mM NaCl, 10%(v/v) glycerol, and 10 mM mercaptoethanol. Stock solutions of inhibitors in ethanol or DMSO. Stock solution of trichostatin A (Sigma): 1 mg/mL in DMSO (3.3 mM). Borate buVer: 6.18 g/L H3BO3 (Merck, Germany), adjusted to pH 9.5 with 1 M NaOH. Naphthalene dicarboxaldehyde (Aldrich, USA): 3.0 mg/mL in methanol. Microplate reader Polarstar Galaxy (BMG Labtechnologies, Germany) with an excitation Wlter of 330 nm and an emission Wlter of 390 nm. Black 96-well microplates (BMG Labtechnologies, Germany).
6.2. Procedure
7.2. Procedure
6. Extraction method with microplate reader based detection 6.1. Materials, buVers, and enzymes
1. Take an aliquot of 24 L of the MAL stock solution and an aliquot of 10 L of the stock solution of Eosin Y and add incubation buVer to a total volume of 1 mL. Dilute the inhibitors with incubation buVer.
1. Dilute 12 L of the MAL stock solution into incubation buVer to a total volume of 1 mL. Dilute the inhibitors with incubation buVer. Dilute trichostatin A to 3.3 M into incubation buVer.
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2. Transfer 50 L of enzyme into each well of a microplate or dilute the enzyme to 50 L with incubation buVer for each sample and each control. For inhibitor screening, add 5 L of diluted inhibitors. Allow to stand for 10 min. Add 5 L of the substrate solution. As a positive control, add 5 L of the trichostatin A solution (3.3 M) as inhibitor. As a negative control, add 5 L of buVer instead of inhibitor. 3. For activity screening, take 50 L of presumable enzyme source or desired quantity and add buVer up to 50 L. As a blank, add 10 L buVer. Use the positive control as standard without enzyme activity and the negative control as standard with maximum enzyme activity. 4. Incubate microplate for desired length of time at 37 °C, usually 90 min (inside the plate reader with heating option or incubation facility with closed lid). 5. Prepare a fresh mixture of 190 L borate buVer, 5 L trichostatin A (3.3 M), and 5 L naphthalene dicarboxaldehyde (3 mg/mL) for each well. Add 200 L of this mixture to each well. 6. Read the plate immediately at 330/390 nm.
8. HPLC assay for sirtuins 8.1. Materials, buVers, and enzymes Fluorescent histone deacetylase substrate ZMAL [19]: 12.6 mM in DMSO, stored at ¡20 °C. Internal standard 7-hydroxycoumarin (Janssen, Germany): 6.3 mg/mL in DMSO. The recombinant sirtuin hSIRT1 was purchased from Biomol (3.5 U/L, Catalogue No. SE-239). Other sirtuins have not been tested so far but might work as well. Incubation buVer: 25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2. A NAD+ stock solution was prepared in water (6 mM). HPLC system: Shimadzu RF 535 Xuorescence detector (330 nm excitation wavelength, 395 nm emission wavelength), Lichrosorb RP-18, 5 m column (150 £ 3 mm, Knauer, Germany) with a guard column of the same material (5 £ 3 mm). Chromatography eluent: acetonitrile/water (40/60 v/v) Xow rate 0.6 mL/min. Retention times are 2.15 min (7-hydroxycoumarin) and 5.40 min (ZMAL). Stop solution: 1 M HCl/0.4 M sodium acetate. 8.2. Procedure 1. Take an aliquot of 12 L of the ZMAL stock solution and an al i quot of 15 L of the stock so lu tion of 7-hydroxycoumarin and add incubation buVer to a total volume of 1 mL. For inhibitor screening, dilute the inhibitors with the incubation buVer to a concentration which is 12-fold higher (for 5 L of inhibitor solution; 60-fold if 1 L of inhibitor solution is added) than the desired assay concentration (dilute at least
1:10, as ethanol and DMSO from the stock solution of the inhibitor interfere with the enzyme). 2. Mix 5 L of the substrate stock solution with 2.5 L of hSIRT1, 5 L of the NAD+ solution, 5 L of the inhibitor solution in the case of inhibitor screening or vehicle as a control (in the case that the inhibitor solution in DMSO is added directly to the reaction volume without further dilution with incubation buVer), and enzyme incubation buVer to a total volume of 60 L. In the case of inhibitors with poor solubility such as sirtinol a solution in pure DMSO may be added but the volume should be limited to 1 L due to background inhibition by DMSO. Incubate at 37 °C for 8 h and stop conversion by the addition of 36 L of stop solution. Extract with 400 L of ethyl acetate and treat consecutively as mentioned in the HPLC extraction method.
9. Homogeneous assay for sirtuins 9.1. Materials, buVers, and enzymes Fluorescent histone deacetylase substrate ZMAL [19] 12.6 mM in DMSO, stock solution of nicotinamide (120 mM in DMSO), both stored at ¡20 °C. The recombinant sirtuin hSIRT1 was purchased from Biomol (3.5 U/L, Catalogue No. SE-239). Incubation buVer: 25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2. Trypsin buVer: Tris– HCl 50 mM, pH 8.0, NaCl 100 mM. A NAD+ stock solution was prepared in water (6 mM). Stock solution of trypsin from bovine pancreas (10,000 BAEE units/ mg protein, Sigma) in trypsin buVer: 6 mg/mL. Inhibitor solution in DMSO or ethanol. Microplate reader Polarstar Galaxy (BMG Labtechnologies, Germany) with an excitation Wlter of 355 or 390 nm and an emission Wlter of 460 nm. Black 96-well microplates (BMG Labtechnologies, Germany). 9.2. Procedure 1. Dilute 10 L of the ZMAL stock solution into incubation buVer to a total volume of 1 mL. Dilute the inhibitors with incubation buVer. 2. Transfer 45 L of a mixture of enzyme and incubation buVer into each well of a microplate for each sample and each control. Generally, 8–16 U of hSirt1 are used. For inhibitor screening, add 5 L of diluted inhibitors. If used inhibitor solutions consist of pure DMSO, a DMSO control is prepared by adding 5 L of DMSO instead of inhibitor solution to the reaction volume. Allow to stand for 10 min on ice. Add 5 L of the substrate solution and 5 L of the NAD+ stock solution. As a negative control, add 5 L of buVer instead of inhibitor.
B. Heltweg et al. / Methods 36 (2005) 332–337
3. For activity screening, take 50 L of presumable enzyme source or desired quantity and add buVer up to 50 L, add 5 L of substrate solution and 5 L of NAD+ stock solution. As a blank, only add 5 L NAD+ stock solution and complement with incubation buVer to 60 L. The negative control (no inhibition, 100 % conversion) is prepared by adding 5 L of NAD+ stock solution to 50 L of the initial reaction mixture. At the end of the incubation time, add 5 L of 7-amino4-methylcoumarine (AMC, 126 M in trypsin buVer) to the reaction volume of the negative control. If desired, the tryptic digest can be controlled by replacing the AMC in the negative control with the same amount of (S)-[5-amino-1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-pentyl]-carbamic acid benzyl ester (ZML). The synthesis of this metabolite is performed starting from Z-(FMOC)Lys-OH similar to the synthesis of ML as outlined elsewhere [14]. 4. Incubate microplate for desired length of time at 37 °C, usually 4 h (inside the plate reader with heating option or incubation facility with closed lid). The amount of enzyme may be reduced by prolongation of the incubation time. 5. Stop solution is prepared by mixing 48 L trypsin buVer, 2 L nicotinamide stock solution, and 10 L trypsin stock solution for each well. Immediately add 60 L of this mixture to each well. 6. Incubate the microplate for another 20 min at 37 °C. 7. Read the plate with a coumarin excitation Wlter (e.g., 355 or 390 nm) and an emission Wlter of 460 nm. The blank subtracted negative control is deWned as 100% conversion and all other conversions are obtained by dividing the blank corrected intensities by that blank corrected intensity. The described assay procedure is not only suitable for sirtuins but also for histone deacetylases class I and II.
false positives when trypsin inhibitors are present in the compound library. Fluorescent compounds may lead to false negatives in the trypsin assay which are lost for the discovery process. 2. The HPLC method is valuable for detecting the resulting metabolite. For routine inhibitor screening, this method cannot be recommended and the homogeneous assay should be performed. Generally, the HPLC method may be used for the veriWcation of the inhibitory activity of Xuorescent compounds. The method without extraction is faster but may lead to increased column contamination by protein fragments. 3. The substrate ZMAL should be preferred to MAL when working with HeLa extracts or sirtuins or when a shorter incubation time is desired (60 instead of 90 min with rat liver extract) for high-throughput screening.
References [1] [2] [3] [4] [5]
[6]
[7] [8] [9] [10]
10. Concluding remarks
[11] [12]
1. The advantage of the homogeneous assay is the speed and the high-throughput suitability. A disadvantage of the NDA detection method may be an increased consumption of enzyme, as blanks and standards should to be run with the same protein content in order to optimize results. For activity tests with limited enzyme sources, especially with varying protein content, the extraction procedure may be the method of choice. Generally, volumes in the homogeneous HDASH assay may be divided by two, so that the enzyme consumption can be kept to a minimum. The NDA detection method does not work with sirtuins and false positives may arise from Xuorescent inhibitors but the latter can be eliminated using the HPLC method. The tryptic digestion method may lead to
337
[13] [14] [15] [16] [17] [18] [19] [20]
[21]
C.M. Grozinger, S.L. Schreiber, Chem. Biol. 9 (2002) 3–16. R.W. Johnstone, Nat. Rev. Drug Discov. 1 (2002) 287–299. S.G. Gray, T.J. Ekstrom, Exp. Cell Res. 262 (2001) 75–83. D. Kölle, G. Brosch, T. Lechner, A. Lusser, P. Loidl, Methods 15 (1998) 323–331. Y. Tanaka, M. Tawaramoto-Sasanuma, S. Kawaguchi, T. Ohta, K. Yoda, H. Kurumizaka, S. Yokoyama, Methods 33 (2004) 3–11. S.J. Darkin-Rattray, A.M. Gurnett, R.W. Myers, P.M. Dulski, T.M. Crumley, J.J. Allocco, C. Cannova, P.T. Meinke, S.L. Colletti, M.A. Bednarek, S.B. Singh, M.A. Goetz, A.W. Dombrowski, J.D. Polishook, D.M. Schmatz, Proc. Natl. Acad. Sci. USA 93 (1996) 13143–13147. J. Taunton, C.A. Hassig, S.L. Schreiber, Science 272 (1996) 408–411. B. Nare, J.J. Allocco, R. Kuningas, S. Galuska, R.W. Myers, M.A. Bednarek, D.M. Schmatz, Anal. Biochem. 267 (1999) 390–396. K. HoVmann, R.M. Söll, A.G. Beck-Sickinger, M. Jung, Bioconj. Chem. 12 (2001) 51–55. D. Wegener, F. Wirsching, D. Riester, A. Schwienhorst, Chem. Biol. 10 (2003) 61–68. D. Wegener, C. Hildmann, D. Riester, A. Schwienhorst, Anal. Biochem. 321 (2003) 202–208. P.A. Marcotte, P.R. Richardson, J. Guo, L.W. Barrett, N. Xu, A. Gunasekera, K.B. Glaser, Anal Biochem 332 (2004) 90–99. K. HoVmann, G. Brosch, P. Loidl, M. Jung, Nucleic Acids Res. 27 (1999) 2057–2058. K. HoVmann, G. Brosch, P. Loidl, M. Jung, Pharmazie 55 (2000) 601–606. K. HoVmann, B. Heltweg, M. Jung, Arch. Pharm. Pharm. Med. Chem. 334 (2001) 248–252. B. Heltweg, M. Jung, Anal. Biochem. 302 (2002) 175–183. B. Heltweg, M. Jung, Arch. Pharm. Pharm. Med. Chem. 335 (2002) 296–300. B. Heltweg, M. Jung, J. Biomol. Screen. 8 (2003) 89–95. B. Heltweg, F. Dequiedt, E. Verdin, M. Jung, Anal. Biochem. 319 (2003) 42–48. B. Heltweg, F. Dequiedt, B.L. Marshall, C. Brauch, M. Yoshida, N. Nishino, E. Verdin, M. Jung, J. Med. Chem. 47 (2004) 5235– 5243. D. Riester, D. Wegener, C. Hildmann, A. Schwienhorst, Biochem. Biophys. Res. Commun. 324 (2004) 1116–1123.
In vitro assays for the determination of histone deacetylase activity Birgit Heltweg a, Johannes Trapp b, Manfred Jung b,¤ a
b
Fred Hutchinson Cancer Research Center, Seattle, USA Department of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany Accepted 30 March 2005
Abstract Histone deacetylases are important regulators of transcription and an emerging target for anticancer drugs. We present an overview over various assay formats that include radiolabelled histones, oligopeptides, and small molecules as substrates. The advantages and disadvantages of the various formats in terms of, e.g., substrate availability, throughput or subtype selectivity are discussed. Detailed procedures for various assay types that can be used for diVerent problems, such as library screening or Xuorescent inhibitor testing, are given. We present a new protocol for a simple high-throughput assay for NAD+-dependent (class III) histone deacetylases, also termed sirtuins. 2005 Elsevier Inc. All rights reserved. Keywords: Histone deacetylase; Sirtuin; Assay; Screening; Aminomethylcoumarin
1. Introduction Histone deacetylases (HDACs) are enzymes that contribute to the posttranslational modiWcations of histones and various non-histone proteins. Deacetylation leads to altered states of conformation and/or activity for the proteins in question [1]. HDACs have been recognized mostly as transcriptional repressors and the aberrant recruitment of deacetylase activity has been linked to distinct leukemia types as a basis for the diseases on a molecular level. Inhibitors of histone deacetylase generally lead to a relief of transcriptional repression which in turn leads to apoptosis and/or diVerentiation in cancer cells. First clinical studies on HDAC inhibitors as new anticancer agents are therefore under way [2]. Eleven subtypes in two classes of those deacetylases that are zinc-dependent amidohydrolases have been discovered so far. There is also a third class of histone deacetylases, also called sirtuins, that requires NAD+ for its catalysis
*
Corresponding author. Fax: +49 761 203 6321. E-mail address: [email protected] (M. Jung).
1046-2023/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2005.03.003
which is not sensitive to class I and II inhibitors [3]. Considering the increasing interest in those enzymes it is very important to have eYcient assays for the detection of their activity at hand. This is needed for both protein isolation and characterization as well as drug screening. We present a comprehensive survey of the existing assays and a new method for the homogeneous measurement of sirtuin activity.
2. Radioactive substrates Traditional assays for the in vitro measurement of histone deacetylase (HDAC) activity involve radioactively labeled histones isolated from chicken reticulocytes or cell culture as the substrates [4]. The substrate properties are diYcult to standardize and show a high batch-to-batch variation. The production of the labeled chicken histones involves pretreatment of the chickens with phenylhydrazine which leads to severe anemia. The animals have to be killed during collection of the cells. The preparation of recombinant histones has recently overcome some of those problems [5]. As an alternative
B. Heltweg et al. / Methods 36 (2005) 332–337
to the histones chemically [3H]-acetylated oligopeptides with 8 [6] or 24 [7] amino acid residues that are derived from histone amino acid sequences have been introduced. They still bear the disadvantages of the use of radioactivity in terms of exposition of personnel and waste management. Additionally, the preparation of those peptides requires sophisticated laboratory equipment or they have to be purchased for a very high price. The deacetylase activity is monitored with both histones and peptides by the extraction of tritiated acetic acid from acidic medium and scintillation counting. This procedure is very time-consuming and therefore not suitable for a high sample throughput. There is a high-throughput version with a biotinylated and tritiated acetyl-histone peptide fragment available that makes use of a scintillation proximity assay [8]. This allows for a large number of samples but the other disadvantages of radioactive oligopeptides are retained.
3. Non-isotopic substrates Oligopeptides that are not radioactively labeled have also been used as in vitro HDAC substrates, e.g., Xuorescein-labeled octapeptides that were monitored by HPLC and Xuorescence detection [9]. Generally, these peptides are rather poor substrates and those methods also have only a low-throughput. They oVer, however, the potential of investigating lysine selectivity for various deacetylases or acetyltransferases. Tripeptides that contain a C-terminal acetyl-lysinecoumarinylamide have been described in a new homogeneous assay [10]. The detection reagent contains trypsin that is able to recognize the oligopeptide as a substrate only if the lysine has been deacetylated. Upon cleavage by trypsin the aminocoumarin is released and can be determined in a homogeneous fashion. A more detailed description concerning its application in high-throughput screening has been reported as well [11]. A similar kit is commercially available for both class I and II, respectively, II enzymes (CycLex). Further Xuorescence and absorption based kits are available by Biomol that work similarily, in part using larger peptidic substrates. A dually labelled sirtuin peptide substrate whose Xuorescent label is internally quenched is cleaved by an endopeptidase to yield a Xuorescent fragment upon deacetylation [12]. Recently, an acetylated lysine derivative, Boc(Ac)LysAMC, also termed MAL, had been introduced as the Wrst non-isotopic substrate for histone deacetylases [13,14]. It is easily accessible by a one-step synthetic procedure and is also commercially available. As the deacetylated metabolite shows the same Xuorescence properties as the parent substrate the conversion was initially monitored by an extraction/HPLC protocol. The use of an internal standard led to increased precision for the extraction proce-
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dure [15]. The procedure was optimized by a new extraction protocol that allowed for a plate reader measurement[16] instead of a HPLC/Xuorescence detection quantiWcation. The original internal standard for that protocol BODIPY 530/550 had to be substituted by Eosin Y [17] as the former is not commercially available anymore. Alternatively, a derivatization reaction can be applied to achieve a homogeneous assay, the so-called HDASH-procedure [18]. In that version, the Xuorescence of the resulting metabolite is quenched by addition of naphthalene dicarboxaldehyde (NDA), an amine detection reagent, and the remaining substrate can be quantitated without separation. MAL can also be determined using the trypsin assay. It is a better substrate for HDAC than the tripeptides but its metabolite is not a very good substrate for trypsin [10]. The diversity of detection protocols allows for an optimized assay depending on the nature of the problem. The HPLC protocols can be applied when the simultaneous detection of substrate and metabolite is desired, when highly Xuorescent compounds should be checked for inhibition or when a plate reader is not available. The extraction/plate reader protocol is used especially when only small amounts of enzyme are available. It is valuable when diVerent samples with diVerent protein contents are screened, e.g., during chromatographic enzyme puriWcation. The homogeneous protocol is best used in inhibitor screening when a high-throughput is desired. Recently, we have introduced Z-(Ac)Lys-AMC, also termed ZMAL [19], as an optimized analogue of MAL. ZMAL is converted by human histone deacetylases faster and to a greater extent, especially valuable is the fact that it is in contrast to MAL a good substrate for the NAD+-dependent class III histone deacetylases (also called sirtuins) that have a completely diVerent mechanism of catalysis [3]. While ZMAL is deacetylated equally well by HDAC1 as a class I deacetylase and by HDAC6 as a class IIa enzyme a moderate subtype selectivity towards HDAC6 was observed for MAL. Similar substrates with a much more pronounced subtype selectivity have been published recently[20,21]. The HPLC and the homogeneous assay have been tested with equimolar amounts of ZMAL instead of MAL and give similar results. The extraction/ plate reader method is expected to behave similarly as well. The NDA detection method does not work with sirtuins. But the deacetylated metabolite of ZMAL, ZML, is a very good substrate for trypsin, and thus, a homogeneous sirtuin assay can be performed with the simple and inexpensive substrate ZMAL (see Table 1).
4. HPLC method without extraction 4.1. Materials, buVers, and enzymes Fluorescent histone deacetylase substrate MAL (Calbiochem): 12.6 mM in DMSO or ethanol, stored at
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Table 1 ClassiWcation of HDAC assays (for details see the corresponding paragraphs of the manuscript) Substrates
Preparation
Commercial availability
Assay and detection
Throughput
Remarks
Radiolabelled histones
From chicken blood, cell culture or recombinant; labelling with [3H]acetyl-CoA and histone acetyltransferase
Components
Extraction and scintillation counting
Low
Native substrate radioactive waste
Histone peptides Unlabelled Radiolabelled
Radiolabelled and biotinylated Fluorescence labelled
Often low reactivity Peptide synthesis Peptide synthesis, [3H]acetyl-CoA and HAT activity or [3H]acetate and chemical labelling Peptide synthesis, biotinylation
Yes Components
HPLC, UV detection Extraction and scintillation counting
Low Low
Components
Scintillation proximity HPLC and UVdetection Peptidase digestion and plate reader Xuorescence
High
Peptide synthesis, labelling Some as a kit
Small molecule Xuorescent substrates Tripeptide-AMCderivatives Lysine-AMCderivatives
Radioactive waste
Low High
Sirtuin substrates available Usually high reactivity
Oligopeptide synthesis
Some as a kit
One to two step syntheses
Components, some as kit
¡20 °C. Internal standard 7-hydroxycoumarin (Janssen, Germany): 6.3 mg/mL in DMSO, stored at ¡20 °C. Histone deacetylase, if not otherwise obtained, from rat liver (see literature [16] for protocol, or commercially available from Calbiochem, USA, or Alexis, Switzerland). Incubation buVer: 1.4 mM NaH2PO4, 18.6 mM Na2HPO4, pH 7.9, 0.25 mM EDTA, 10 mM NaCl, 10% (v/v) glycerol, and 10 mM mercaptoethanol. Stop mixture: 1 M HCl/0.4 M sodium acetate. HPLC system with a Shimadzu RF 535 Xuorescence detector (excitation wavelength 330 nm, emission wavelength 395 nm) and a Multospher RP-18, 5 m column (250 £ 4 mm, CS-Chromatographie, Germany) with a guard column of the same material (5 £ 4 mm). Chromatography eluent: acetonitrile/water/triXuoroacetic acid (55/45/0.01 v/v). Stock solutions of inhibitors in DMSO or methanol/ethanol.
2.
3.
4.
4.2. Procedure 5. 1. Take an aliquot of 12 L of the MAL stock solution and an al i quot of 15 L of the stock so lu tion of 7-hydroxycoumarin and add incubation buVer to a total volume of 1 mL. For inhibitor screening, dilute
6. 7.
plate reader Xuorescence HPLC and Xuorescence detection or extraction and plate reader Xuorescence
High
Tryptic or chemical detection, plate reader Xuorescence
High
Lowmedium
Sirtuin substrates and HDAC class I and II selective substrates available, components much cheaper than kits
the inhibitors with the incubation buVer to a concentration which is 12-fold higher than the desired assay concentration (dilute at least 1:10, as ethanol and DMSO from the stock solution of the inhibitor interfere with the enzyme). For standards without enzyme activity, take 110 L incubation buVer, add 10 L of the substrate/internal standard solution and mix well. Chill on ice. For inhibitor samples, take 100 L of enzyme or dilute the enzyme to 100 L with incubation buVer for each sample. Add 10 L diluted inhibitors and mix gently. Keep on ice for 10 min. Add 10 L of the substrate/internal standard solution and mix gently. Allow to stand at 4 °C for another 15 min. As enzyme activity standards, take 100 L of enzyme or diluted enzyme and add 10 L of the buVer. Keep on ice for 10 min. Add 10 L of the substrate/internal standard solution and mix gently. Allow to stand at 4 °C for another 15 min. Incubate the samples and standards for the desired length of time at 37 °C, usually 90 min. Stop the reaction by addition of 1000 L of acetonitrile and vortex approximately 30 s. Centrifuge at 10,000 rpm for 5 min.
B. Heltweg et al. / Methods 36 (2005) 332–337
8. Take 600 L of the supernatant and inject 20 L onto the HPLC system (Xow rate 0.5 mL/min). Retention time is 5.06 min for the deacetylated substrate, 5.64 min for the internal standard, and 7.09 min for the substrate.
5. HPLC extraction method 5.1. Materials, buVers, and enzymes Stop mixture: 1 M HCl/0.4 M sodium acetate. HPLC system: Shimadzu RF 535 Xuorescence detector (330 nm excitation wavelength, 395 nm emission wavelength), Lichrosorb RP-18, 5 m column (150 £ 3 mm, Knauer, Germany) with a guard column of the same material (5 £ 3 mm). Chromatography eluent: acetonitrile/water (40/60 v/v). 5.2. Procedure 1. Prepare samples and standards and incubate at 37 °C as described in HPLC method without extraction. 2. Stop the reaction by addition of 72 L of stop mixture and mix well. 3. Add 800 L ethyl acetate and vortex approximately 30 s. 4. Centrifuge at 10,000 rpm for 5 min. 5. Take 200 L of the upper phase and remove ethyl acetate by a stream of nitrogen. 6. Dissolve residue in 600 L of the chromatography eluent, mix well, and inject 20 L onto the HPLC system (Xow rate 0.6 mL/min). Retention time of the internal standard is 2.17 and 3.47 min for the substrate.
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2. For standards without enzyme activity, take 110 L of incubation buVer, add 10 L of the substrate/internal standard solution, and mix well. Chill on ice. 3. For inhibitor samples, take 100 L of enzyme or dilute the enzyme to a total volume of 100 L with incubation buVer for each sample. Add 10 L of diluted inhibitors and mix gently. Keep on ice for 10 min. Add 10 L of the substrate/internal standard solution and mix gently. Keep on ice for another 15 min. 4. For enzyme activity standards, take 100 L of enzyme or diluted enzyme and add 10 L of incubation buVer. Keep on ice for 10 min. Add 10 L of the substrate/internal standard solution and mix gently. Keep on ice for another 15 min. 5. Incubate samples and standards for desired length of time at 37 °C, usually 90 min. 6. Stop the reaction by addition of 400 L of 1 M HCl and mix well. 7. Add 800 L ethyl acetate and vortex approximately 30 s. 8. Centrifuge at 10,000 rpm for 5 min. 9. Take 400 L of upper phase and evaporate ethyl acetate by a stream of nitrogen. 10. Dissolve residue in 600 L acetonitrile/buVer mixture, vortex, and transfer 250 L into microplate. Read the plate at 330/390 nm.
7. Homogeneous assay for class I and II deacetylases (HDASH) 7.1. Materials, buVers, and enzymes
Internal standard Eosin Y (Merck, Germany): 9.3 mg/mL in ethanol. Microplate reader Polarstar Galaxy (BMG Labtechnologies, Germany) with an excitation Wlter of 330 nm and an emission Wlter of 390 nm. Black 96-well microplates (BMG Labtechnologies, Germany). Solvent: 1 part of buVer (pH 8, 0.5 mM KH2PO4, 0.46 mM NaOH) is diluted with 59.4 parts of water and this is then mixed with 39.6 parts of acetonitrile.
Histone deacetylase in phosphate buVer or another buVer (e.g., Hepes) without primary amines (do not use tris buVer!). Incubation buVer: 1.4 mM NaH2PO4, 18.6 mM Na2HPO4, pH 7.9, 0.25 mM EDTA, 10 mM NaCl, 10%(v/v) glycerol, and 10 mM mercaptoethanol. Stock solutions of inhibitors in ethanol or DMSO. Stock solution of trichostatin A (Sigma): 1 mg/mL in DMSO (3.3 mM). Borate buVer: 6.18 g/L H3BO3 (Merck, Germany), adjusted to pH 9.5 with 1 M NaOH. Naphthalene dicarboxaldehyde (Aldrich, USA): 3.0 mg/mL in methanol. Microplate reader Polarstar Galaxy (BMG Labtechnologies, Germany) with an excitation Wlter of 330 nm and an emission Wlter of 390 nm. Black 96-well microplates (BMG Labtechnologies, Germany).
6.2. Procedure
7.2. Procedure
6. Extraction method with microplate reader based detection 6.1. Materials, buVers, and enzymes
1. Take an aliquot of 24 L of the MAL stock solution and an aliquot of 10 L of the stock solution of Eosin Y and add incubation buVer to a total volume of 1 mL. Dilute the inhibitors with incubation buVer.
1. Dilute 12 L of the MAL stock solution into incubation buVer to a total volume of 1 mL. Dilute the inhibitors with incubation buVer. Dilute trichostatin A to 3.3 M into incubation buVer.
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2. Transfer 50 L of enzyme into each well of a microplate or dilute the enzyme to 50 L with incubation buVer for each sample and each control. For inhibitor screening, add 5 L of diluted inhibitors. Allow to stand for 10 min. Add 5 L of the substrate solution. As a positive control, add 5 L of the trichostatin A solution (3.3 M) as inhibitor. As a negative control, add 5 L of buVer instead of inhibitor. 3. For activity screening, take 50 L of presumable enzyme source or desired quantity and add buVer up to 50 L. As a blank, add 10 L buVer. Use the positive control as standard without enzyme activity and the negative control as standard with maximum enzyme activity. 4. Incubate microplate for desired length of time at 37 °C, usually 90 min (inside the plate reader with heating option or incubation facility with closed lid). 5. Prepare a fresh mixture of 190 L borate buVer, 5 L trichostatin A (3.3 M), and 5 L naphthalene dicarboxaldehyde (3 mg/mL) for each well. Add 200 L of this mixture to each well. 6. Read the plate immediately at 330/390 nm.
8. HPLC assay for sirtuins 8.1. Materials, buVers, and enzymes Fluorescent histone deacetylase substrate ZMAL [19]: 12.6 mM in DMSO, stored at ¡20 °C. Internal standard 7-hydroxycoumarin (Janssen, Germany): 6.3 mg/mL in DMSO. The recombinant sirtuin hSIRT1 was purchased from Biomol (3.5 U/L, Catalogue No. SE-239). Other sirtuins have not been tested so far but might work as well. Incubation buVer: 25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2. A NAD+ stock solution was prepared in water (6 mM). HPLC system: Shimadzu RF 535 Xuorescence detector (330 nm excitation wavelength, 395 nm emission wavelength), Lichrosorb RP-18, 5 m column (150 £ 3 mm, Knauer, Germany) with a guard column of the same material (5 £ 3 mm). Chromatography eluent: acetonitrile/water (40/60 v/v) Xow rate 0.6 mL/min. Retention times are 2.15 min (7-hydroxycoumarin) and 5.40 min (ZMAL). Stop solution: 1 M HCl/0.4 M sodium acetate. 8.2. Procedure 1. Take an aliquot of 12 L of the ZMAL stock solution and an al i quot of 15 L of the stock so lu tion of 7-hydroxycoumarin and add incubation buVer to a total volume of 1 mL. For inhibitor screening, dilute the inhibitors with the incubation buVer to a concentration which is 12-fold higher (for 5 L of inhibitor solution; 60-fold if 1 L of inhibitor solution is added) than the desired assay concentration (dilute at least
1:10, as ethanol and DMSO from the stock solution of the inhibitor interfere with the enzyme). 2. Mix 5 L of the substrate stock solution with 2.5 L of hSIRT1, 5 L of the NAD+ solution, 5 L of the inhibitor solution in the case of inhibitor screening or vehicle as a control (in the case that the inhibitor solution in DMSO is added directly to the reaction volume without further dilution with incubation buVer), and enzyme incubation buVer to a total volume of 60 L. In the case of inhibitors with poor solubility such as sirtinol a solution in pure DMSO may be added but the volume should be limited to 1 L due to background inhibition by DMSO. Incubate at 37 °C for 8 h and stop conversion by the addition of 36 L of stop solution. Extract with 400 L of ethyl acetate and treat consecutively as mentioned in the HPLC extraction method.
9. Homogeneous assay for sirtuins 9.1. Materials, buVers, and enzymes Fluorescent histone deacetylase substrate ZMAL [19] 12.6 mM in DMSO, stock solution of nicotinamide (120 mM in DMSO), both stored at ¡20 °C. The recombinant sirtuin hSIRT1 was purchased from Biomol (3.5 U/L, Catalogue No. SE-239). Incubation buVer: 25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2. Trypsin buVer: Tris– HCl 50 mM, pH 8.0, NaCl 100 mM. A NAD+ stock solution was prepared in water (6 mM). Stock solution of trypsin from bovine pancreas (10,000 BAEE units/ mg protein, Sigma) in trypsin buVer: 6 mg/mL. Inhibitor solution in DMSO or ethanol. Microplate reader Polarstar Galaxy (BMG Labtechnologies, Germany) with an excitation Wlter of 355 or 390 nm and an emission Wlter of 460 nm. Black 96-well microplates (BMG Labtechnologies, Germany). 9.2. Procedure 1. Dilute 10 L of the ZMAL stock solution into incubation buVer to a total volume of 1 mL. Dilute the inhibitors with incubation buVer. 2. Transfer 45 L of a mixture of enzyme and incubation buVer into each well of a microplate for each sample and each control. Generally, 8–16 U of hSirt1 are used. For inhibitor screening, add 5 L of diluted inhibitors. If used inhibitor solutions consist of pure DMSO, a DMSO control is prepared by adding 5 L of DMSO instead of inhibitor solution to the reaction volume. Allow to stand for 10 min on ice. Add 5 L of the substrate solution and 5 L of the NAD+ stock solution. As a negative control, add 5 L of buVer instead of inhibitor.
B. Heltweg et al. / Methods 36 (2005) 332–337
3. For activity screening, take 50 L of presumable enzyme source or desired quantity and add buVer up to 50 L, add 5 L of substrate solution and 5 L of NAD+ stock solution. As a blank, only add 5 L NAD+ stock solution and complement with incubation buVer to 60 L. The negative control (no inhibition, 100 % conversion) is prepared by adding 5 L of NAD+ stock solution to 50 L of the initial reaction mixture. At the end of the incubation time, add 5 L of 7-amino4-methylcoumarine (AMC, 126 M in trypsin buVer) to the reaction volume of the negative control. If desired, the tryptic digest can be controlled by replacing the AMC in the negative control with the same amount of (S)-[5-amino-1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-pentyl]-carbamic acid benzyl ester (ZML). The synthesis of this metabolite is performed starting from Z-(FMOC)Lys-OH similar to the synthesis of ML as outlined elsewhere [14]. 4. Incubate microplate for desired length of time at 37 °C, usually 4 h (inside the plate reader with heating option or incubation facility with closed lid). The amount of enzyme may be reduced by prolongation of the incubation time. 5. Stop solution is prepared by mixing 48 L trypsin buVer, 2 L nicotinamide stock solution, and 10 L trypsin stock solution for each well. Immediately add 60 L of this mixture to each well. 6. Incubate the microplate for another 20 min at 37 °C. 7. Read the plate with a coumarin excitation Wlter (e.g., 355 or 390 nm) and an emission Wlter of 460 nm. The blank subtracted negative control is deWned as 100% conversion and all other conversions are obtained by dividing the blank corrected intensities by that blank corrected intensity. The described assay procedure is not only suitable for sirtuins but also for histone deacetylases class I and II.
false positives when trypsin inhibitors are present in the compound library. Fluorescent compounds may lead to false negatives in the trypsin assay which are lost for the discovery process. 2. The HPLC method is valuable for detecting the resulting metabolite. For routine inhibitor screening, this method cannot be recommended and the homogeneous assay should be performed. Generally, the HPLC method may be used for the veriWcation of the inhibitory activity of Xuorescent compounds. The method without extraction is faster but may lead to increased column contamination by protein fragments. 3. The substrate ZMAL should be preferred to MAL when working with HeLa extracts or sirtuins or when a shorter incubation time is desired (60 instead of 90 min with rat liver extract) for high-throughput screening.
References [1] [2] [3] [4] [5]
[6]
[7] [8] [9] [10]
10. Concluding remarks
[11] [12]
1. The advantage of the homogeneous assay is the speed and the high-throughput suitability. A disadvantage of the NDA detection method may be an increased consumption of enzyme, as blanks and standards should to be run with the same protein content in order to optimize results. For activity tests with limited enzyme sources, especially with varying protein content, the extraction procedure may be the method of choice. Generally, volumes in the homogeneous HDASH assay may be divided by two, so that the enzyme consumption can be kept to a minimum. The NDA detection method does not work with sirtuins and false positives may arise from Xuorescent inhibitors but the latter can be eliminated using the HPLC method. The tryptic digestion method may lead to
337
[13] [14] [15] [16] [17] [18] [19] [20]
[21]
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