An ATPase Assay Using Scintillation Proximity Beads for High-Throughput Screening or Kinetic Analysis

Analytical Biochemistry 304, 55– 62 (2002) doi:10.1006/abio.2002.5632, available online at http://www.idealibrary.com on

An ATPase Assay Using Scintillation Proximity Beads for High-Throughput Screening or Kinetic Analysis Jamie A. Jeffery, Jeffrey R. Sharom, Monika Fazekas, Penny Rudd, Ewald Welchner, Louise Thauvette, and Peter W. White 1 Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard St., Laval, Quebec, Canada H7S 2G5

Received October 25, 2001; published online April 3, 2002

A new procedure for measuring ATPase activity in which ␥- 33P-labeled inorganic orthophoshate is detected by addition of ammonium molybdate followed by selective adsorption of the resulting phosphomolybdate to scintillation proximity beads in the presence of cesium chloride is described. This method is shown to give accurate and reproducible results over a wide range of detection conditions and product concentrations. It requires no separation or filtration steps and is highly compatible with automated highthroughput screening. Rates of hydrolysis are easily and accurately determined over a wide range, and thus the method is useful for kinetic studies also. We show that this scintillation proximity assay is useful for the study of the E1 helicase of human papillomavirus, but it is a general procedure which could also be applied to any ATPase or other nucleotide triphosphate-hydrolyzing enzyme or any other enzyme which generates orthophosphate as a reaction product. © 2002 Elsevier Science (USA)

ATP hydrolysis provides cells with energy for many activities, including membrane transport, movement of flagella in bacteria, myofibril contraction in muscle cells, and translocation of proteins on DNA. Consequently, a number of different methods have been reported for the measurement of ATPase activity. In our own work, we have been interested in the ATP-dependent DNA-unwinding activity of the E1 helicase from human papillomavirus (HPV) 2 types 6 and 11 (1). 1 To whom correspondence should be addressed. Fax: (450) 6824642. E-mail: [email protected]. 2 Abbreviations used: AmMo, ammonium molybdate; ATP-␥-S, adenosine-5⬘-O-(3-thio)triphosphate; dpm, decays per minute; DTT, dithiothreitol; HPV, human papillomavirus; MgOAc, magnesium acetate; PVT, polyvinyltoluene; SPA, scintillation proximity assay.

0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

There are approximately 100 HPV types, some of which are the etiological agents of anogenital cancers, most notably cervical cancer, or of genital warts. The virus infects basal epithelial cells, and viral replication is linked to the differentiation of the infected cells (2). Replication of the viral genome requires only two viral proteins, known as E1 and E2 (3, 4). E2 is a sequencespecific DNA-binding protein which helps recruit E1 to the viral origin of replication (5). E1 itself is a helicase (6). It assembles into hexamers at the origin, recruits cellular proteins to form an active replication complex, and unwinds DNA at the replication fork (7). Both the initial assembly process (8, 9) and the DNA unwinding (6) are dependent on E1-catalyzed ATP hydrolysis. Because viral replication is dependent on E1 ATPase activity, inhibitors could potentially be useful as antiviral drugs (10). We wished to identify an assay method for E1 compatible with high-throughput compound screening. E1 has a K m for ATP of approximately 10 ␮M, so to work in conditions sensitive to ATP-competitive inhibitors, we wanted to use low micromolar concentrations of ATP. Thus, the more common colorimetric methods for assaying ATPase activity in the 96-well plate format were not sufficiently sensitive. Very sensitive ATPase assays using radiolabeled ATP have been developed, but these methods rely on the separation of labeled product from unreacted substrate, usually by TLC on polyethyleneimine-coated cellulose (11). We used this method for initial characterization of E1 activity, but it is not as convenient as a screening assay. Another radioactive method relies on the selective adsorption of ATP to charcoal: reactions can be filtered through charcoal and the filtrate counted to determine the amount of reaction product (12, 13). This method may offer improved throughput relative to TLC, but procedures requiring filtration are not optimal for automated screening. 55

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Many variations on a method in which inorganic orthophosphate (P i) is combined with ammonium molybdate (AmMo) under acidic conditions to yield anionic phosphomolybdate ([PMo 12O 40] 3⫺) have been reported. The anion itself is yellow, while the reduced form has a more intense blue color; in either case colorimetric detection is possible (14). Improved sensitivity can be obtained by adding certain cationic dyes, most commonly malachite green, the absorbance of which increases on formation of a complex with phosphomolybdate at low pH (15). Background due to acid hydrolysis of ATP can be reduced by adding citrate, shortly after ammonium molybdate, to chelate any P i generated by nonenzymatic hydrolysis (16). This format is potentially suitable for screening. Optimized versions are able to detect phosphate concentrations as low as 100 nM but only if relatively large volumes (e.g., 1 ml) are used (17). The phosphomolybdate anion has a ⫺3 charge spread over 53 atoms and is in fact relatively hydrophobic. It has therefore been found that ATPase activity can be monitored by adding ammonium molybdate to assays with radiolabeled P i and then extracting the radiolabeled phosphomolybdate complex into organic solvents (18) or adsorbing it onto polyvinylpolypyrrolidone (19). These methods cannot be easily adapted to 96-well plate format, however, and thus cannot be used in modern high-throughput screening laboratories. For many enzymes or receptors, high-throughput assays using radioactivity are commonly performed using polyvinyltoluene (PVT) beads impregnated with scintillant (20). Scintillation proximity assays (SPA) are configured such that either the substrate or the product (or one of two partners in a binding assay) binds specifically to the beads by virtue of an affinity coating such as streptavidin, protein A, or glutathione. Only bound radioactive species generate a significant scintillation signal, so a physical separation step is unnecessary. We reasoned that the surface of PVT SPA beads might be sufficiently hydrophobic to bind selectively to phosphomolybdate even in the presence of excess ATP. As described below, this principle does provide the basis for an assay which is straightforward, accurate, and highly reproducible (21). The assay signal is linear over a wide P i concentration range and is thus very useful for steady-state kinetic studies. Although developed to monitor ATPase activity of the HPV E1 helicase, this method should be applicable to any ATPase or in fact to any enzyme which generates inorganic orthophosphate, if the appropriate radiolabeled substrate can be obtained. MATERIALS AND METHODS

Materials. HPV6 E1, 11 E1, and the truncated HPV11 E1(72-649) were expressed from baculovirusinfected insect cells and purified as described (1). Am-

monium molybdate was obtained from A & C American Chemicals, Ltd. [␥- 33P]ATP (3000 Ci/mmol) was obtained from Perkin–Elmer/NEN or Amersham Pharmacia Biotech; lower blanks were obtained using material shipped on dry ice. Uncoated (Product RPQ0542) or streptavidin-coated (Product RPNQ0007) SPA beads were obtained from Amersham Pharmacia Biotech. Other reagents were obtained from Sigma. Enzymatic reactions. [␥- 33P]ATP was diluted on receipt 100-fold in reaction buffer and stored in small aliquots at ⫺80°C. Fresh substrate was obtained each month. Typical assays were carried out for 2 h at room temperature in 96-well Optiplates (Packard) in a total volume of 40 or 45 ␮l. The reaction buffer consisted of 20 mM Hepes, pH 7.5, 1 mM DTT, 0.05 mM EDTA, and 0.005% (v/v) IGEPAL CA-630 detergent. The detergent is not required for detection, but slightly enhanced signals for E1 assays. Two percent Me 2SO was included in some experiments and had no effect on signal. Standard assays contained 500 ␮M MgOAc, 1 ␮M ATP, and 30 nCi/well [␥- 33P]ATP. Sufficient E1 helicase was used to give approximately 20 –30% hydrolysis after 2 h. In some cases, larger reactions were carried out in microfuge tubes or polypropylene plates and then 45-␮l aliquots were transferred to 96-well reaction plates for detection. For 100% hydrolysis reactions, higher concentrations of enzyme were used to give complete hydrolysis of substrate within 2 h. Detection by TLC. For TLC detection, reactions performed as described above were stopped by adding one-half volume of cold EDTA (500 mM). One-microliter aliquots were spotted onto polyethyleneimine cellulose TLC plates (Sigma), typically 12 spots per each half plate. Spots were allowed to dry, and samples were chromatographed in a running buffer of 1 M formic acid/1 M LiCl. Plates were then dried and exposed on a storage phosphor screen, and the resulting signal was quantified using a STORM 860 imaging system (Amersham Pharmacia Biotech/Molecular Dynamics). Signal obtained from no-enzyme blank reactions run in parallel, typically 1– 4%, was subtracted. Detection by scintillation proximity. AmMo was dissolved to 20 mg/ml in 2.4 M HCl, and PVT SPA beads (coated with streptavidin or uncoated) were suspended at a concentration of 15 mg/ml in a buffer consisting of 50 mM Hepes, pH 7.5, plus 0.02% sodium azide. The AmMo solution and bead suspension were premixed in a ratio of 1:2 and enzymatic reactions were stopped by the addition of 40 ␮l of this mixture, followed within 2 min by the addition of 80 ␮l 7 M cesium chloride containing 0.1 M citric acid. In some cases, the AmMo solution and bead suspension were added sequentially with no difference in results. Signals were detected by scintillation counting using a TopCount NXT microplate scintillation counter (Perkin–Elmer/ Packard Instruments) and values for no-enzyme

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FIG. 1. Schematic representation of the scintillation proximity ATPase assay. (A) Hydrolysis of ATP to generate radioactive P i. (B) Addition of polyvinyltoluene scintillation proximity beads (PVT-SPA beads) suspended in a solution of ammonium molybdate and HCl. The phosphomolybdate anion (PMo 6O 40) 3⫺ (small red circles) forms and then adsorbs to the surface of SPA beads (larger gray circles). (C) Addition of CsCl to float the beads.

blank reactions run in parallel, typically 1– 4%, were subtracted. Reactions turned a very faint yellow and produced a small amount of very fine precipitate on the addition of the ammonium molybdate/bead mixture (or the AmMo solution alone, prior to the separate addition of beads). The color change and precipitation were found to be dependent on the presence of DTT and did not affect results. Variations on this standard procedure are described in the text or figure legends. Initial velocity kinetics. For reactions run to determine the value of K i (ATP-␥-S), cold and radiolabeled ATP were maintained at a constant ratio (typically 100 ␮Ci per ␮mol of unlabeled ATP), and the total magnesium concentration was adjusted to give a constant excess, usually 500 ␮M. At low substrate concentrations, excess magnesium is needed to ensure quantitative formation of the ATP–magnesium complex, which has a K d value of approximately 50 ␮M (22). Aliquots were taken from triplicate reactions at multiple time points between 20 and 120 min, and signal was detected by scintillation proximity and in some cases by TLC, as described under Results. Observed rates were calculated using data at 20% conversion or lower to maintain initial rate conditions. Kinetic data were analyzed using the appropriate equations in the program GraFit 3.0 (Erithicus Software Ltd.). RESULTS

Basic conditions for detection of orthophosphate by scintillation proximity. A schematic representation of the assay is shown in Fig. 1. Reactions are carried out using unlabeled ATP with a trace amount of

[␥- 33P]ATP. For assays dependent on scintillation proximity, it is important to use 33P-labeled substrates, since the high-energy ␤-particles emitted by 32P would be captured by the beads even for unbound species, and thus no specific signal would be observed. Subsequent to enzymatic hydrolysis, an acidic solution of ammonium molybdate is added, resulting in the formation of phosphmolybdate as for colorimetric assays using malachite green. Due to the hydrophobicity of this complex, it is selectively adsorbed to the polyvinyltoluene SPA bead surface, while unhydrolyzed ATP remains in solution. Precoated streptavidin beads were used in many of our experiments; however, we found that a number of other PVT SPA bead coatings (e.g., wheatgerm, protein A, or glutathione) performed equivalently, and in fact it is now possible to obtain uncoated beads and we found that these could be used interchangeably. In a final step prior to detection, a cesium chloride solution is added. This increases the density of the solution such that the beads, which are only slightly more dense that pure water (specific gravity ⫽ 1.05 mg/mL), float. The resulting concentration of beads at the surface significantly improves scintillation proximity signals for 33P. The concomitant increase in ionic strength probably also enhances the hydrophobic interaction between phosphomolybdate and beads. After floatation (which occurs over approximately 1 h), assays can be counted using a microplate scintillation counter. No physical separation of substrate and product is necessary. We typically carry out 40-␮l reactions using 1 ␮M total ATP containing 30 nCi of [␥- 33P]ATP. Final concentrations of ammonium molybdate and HCl are similar to those reported for colorimetric assays (16). In initial experiments, AmMo and SPA beads were added

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separately, but we found that the two components could be premixed and added together with no effect on signal. In fact, this mixture could be made several hours in advance, or even the previous day if kept at 4°C (data not shown). A final CsCl concentration of at least 1 M was needed to efficiently float beads, and higher concentrations of up to 3.5 M gave improved results. We found it best to add the CsCl solution immediately after the AmMo and then to read plates after 1 h. Originally, longer incubation resulted in a steady increase in background due to the nonenzymatic hydrolysis of ATP under the acidic detection conditions (data not shown). Thus, as previously reported for assays using malachite green (16), an important modification was to add citric acid, with the CsCl, to trap excess uncomplexed molybdate prior to nonenzymatic hydrolysis of ATP. In the presence of citric acid the SPA signal was stable for at least 24 h (see below), indicating that neither complexation of P i formed nonenzymatically nor decomposition of phosphomolybdate formed prior to addition of citric acid was significant. 3 A significant background signal was consistently observed in these experiments and could in principle be due to several sources. TLC detection showed that a small amount of P i (1–5%) was present in commercial preparations of radiolabeled ATP. In addition, it was also possible that unhydrolyzed ATP itself might weakly associate with SPA beads. Finally, SPA beads could capture some ␤ particles from unbound radiolabeled ATP. In the experiment shown in Fig. 2, samples of ATP either incubated without enzyme or hydrolyzed completely by the addition of excess E1 were diluted to give a concentration range of 0.5 to 10 ␮M. Detection was performed with or without the addition of AmMo. All signals were found to be linear over the concentration range tested. The signal obtained for reaction blanks in the presence of AmMo was roughly 10-fold higher than that from either hydrolyzed or nonhydrolyzed ATP in the absence of AmMo. This dependence on AmMo indicates that all but about 10% of the background is due to preexisting P i in the radiolabeled ATP. To minimize background, we obtain the freshest material possible, diluting it on receipt for storage at ⫺80°C. The residual background observed in the absence of AmMo is probably derived mostly from the capture of ␤ particles emitted from unbound rather than nonspecifically bound species, since one might expect some 3 Though citrate has a qualitatively similar effect in both colorimetric and SPA formats, a reviewer has pointed out that for colorimetric assays one usually observes an increase in background after several hours even in the presence of citrate. We have also found this to be true; however, in this scintillation proximity procedure we see no significant change in blank signal over time (see Fig. 4). Final concentrations of both AmMo and citrate in both procedures are similar (16), but it is possible that this difference is due to the much lower levels of ATP used here, typically 1–100 ␮M, compared to concentrations often greater than 1 mM in colorimetric assays.

FIG. 2. Linearity of assay signal and background. A reaction with 10 ␮M ATP and 500 ␮M MgOAc was hydrolyzed to completion using an excess of E1; a similar mixture lacking E1 (reaction blank) was also prepared. Following hydrolysis, both mixtures were diluted in varying proportions with reaction buffer plus 500 ␮M MgOAc, to give a final range from 0.5 to 10 ␮M. Solutions were distributed in 96-well plates, 40 ␮l per well (30 to 600 nCi), and detection was performed by adding 40 ␮l of SPA bead suspension (10 mg/ml) in 0.8 M HCl with or without 0.67% AmMo, followed by 80 ␮l of 7 M CsCl with 0.2 M citric acid. Data are shown for reactions with AmMo (F), reaction blanks with AmMo (■), reactions without AmMo (E), and reaction blanks without AmMo (䊐). Data are plotted on a log–log scale.

difference in the affinity of ATP and P i for the PVT bead surface (compare open squares and circles in Fig. 2). Application of the scintillation proximity method to compound screening. The ATPase SPA should be well suited to automated high-throughput screening, and Fig. 3 shows an example of the results expected under screening conditions. Plotted are the data for a large number of blanks and ATPase reactions in the presence or absence of 3 ␮M ATP-␥-S, a known inhibitor which closely resembles the substrate. To retain maximum sensitivity to competitive inhibitors, reactions were run using 1 ␮M ATP, well below K m (1), and conversion was kept low to avoid depleting the substrate. In this experiment, the signal to background ratio was 10 and the z⬘ statistic (23) was 0.84. The z⬘ value is a measure of the performance of an assay which takes into account both the difference between signal and background and the standard deviations on these. Values can vary from 1.0 to below zero, and any value above 0.5 is considered very acceptable for screening. The average level of inhibition at this concentration of ATP-␥-S was 30% and is clearly distinguished from control reactions. In screening applications, it is often observed that colored compounds partially quench the scintillation signal emitted by SPA beads. However, most colored compounds are bleached at the low pH used here for detection, and the floatation of beads minimizes the effect of remaining quencher by reducing the volume of solution through which the signal must pass. Thus color quenching is unlikely to be a significant issue for this method. For a robust screening assay, absolute signal and measured inhibition should be relatively insensitive to small variations in detection conditions, which might

SCINTILLATION PROXIMITY ATPase ASSAY

FIG. 3. Reproducibility of the assay signal under screening conditions. Reaction blanks ({) and reactions in the absence (F) or presence (Œ) of 3 ␮M ATP-␥-S were each run in 24 wells of three 96-well plates. The assay was run for 2 h at room temperature using 3.5 nM HPV6 E1, 1 ␮M ATP, and 500 ␮M MgOAc (45 ␮l per well). Signals were detected as described under Materials and Methods. Plates were counted 5 h after addition of CsCl. The average signal for the control reactions was 4050 ⫾ 170 cpm, corresponding to 14 ⫾ 0.7% conversion, whereas the background was 390 ⫾ 25 cpm or 1.5 ⫾ 0.1%. The average signal from wells containing ATP-␥-S was 2941 ⫾ 95 cpm or 11 ⫾ 0.4% conversion. Values of percentage conversion are calculated from reactions run with a high concentration of enzyme, confirmed to give 100% conversion by TLC, and are given after background subtraction.

occur from one day to the next. To test this for the ATPase SPA, a number of reactions were run in parallel, with or without the inhibitor ATP-␥-S, and then detected using either standard conditions or twofold variations in HCl, AmMo, or citric acid concentrations (Fig. 4). Reaction plates were also read at three different times, from 1.5 to 20 h after addition of CsCl. There was no significant variation of signal over this time for either samples or blanks, and in other experiments only a small loss of signal was observed after several days (data not shown). Although changes in detection conditions or timing led to slight variation in the signal measured and consequently in the signal to background ratio (from 15 to 26), there was almost no difference in measured inhibition, which varied only between 65.9 and 68.5%. One would expect day-to-day differences in solution composition to be much smaller than the range tested here, and thus the scintillation proximity method should give highly reproducible results in the screening environment. Application of scintillation proximity detection to kinetic experiments. Although the SPA detection procedure is well-suited to screening assays run at very low concentrations of ATP, it was not initially clear whether it could be adapted to mechanistic studies carried out over a wide range of substrate concentrations. In reactions at higher substrate, only a few percent of the substrate would be converted to product, and this low conversion rate might be difficult to measure accurately given that the starting material already contains a similar level of P i. Furthermore, since we had observed that the assay signal varied with SPA bead concentration, it seemed likely that at higher

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FIG. 4. Variation of signal, background, and observed inhibition under different detection conditions. Reactions (30 ␮l) were run for 1 h at room temperature using 1 ␮M ATP, 500 ␮M MgOAc, and approximately 10 nM HPV11 E1, to give approximately 35% conversion. Detection was performed using 30 ␮l of AmMo in HCl, 30 ␮l of SPA beads at 15 mg/ml, and 90 ␮l of 7 M CsCl plus citric acid. Average signals obtained for reaction blanks (black), reactions containing E1 10 ␮M ATP-␥-S (dark gray), and reactions with E1 alone (light gray) are shown at 1.5, 6.5, or 20 h (from left to right within each set) for detection conditions A–G. Error bars correspond to the standard deviation on triplicate reactions. For A, solutions of 2% AmMo in 1.2 M HCl and 0.2 M citric acid in 7 M CsCl were used. In B and C half and twice (respectively) the HCl concentration was used, in D and E half and twice (respectively) the AmMo concentration was used, and in F and G half and twice (respectively) the citric acid concentration was used.

substrate concentrations the phosphomolybdate binding capacity of the beads might be saturated and signal would no longer be linear with respect to the concentration of product. Interestingly, this does not seem to be the case. For the experiment in Fig. 5, ATP was hydrolyzed and serially diluted; then P i product was

FIG. 5. Phosphomolybdate binding capacity of streptavidin SPA beads. A substrate mixture containing 200 ␮M ATP in 700 ␮M MgOAc was hydrolyzed to completion (verified by TLC) using an excess of E1(72-649) (200 nM) and then diluted with buffer containing 500 ␮M MgOAc to give final P i concentrations ranging from 200 ␮M to 200 nM. Detection was performed by adding 40 ␮l premixed AmMo-HCl-SPA beads to 40 ␮l of diluted reaction mixture, followed by 80 ␮l of CsCl/citric acid. Bead concentrations in the premixed suspensions were 5 mg/ml (}), 10 mg/ml (■), 20 mg/ml (Œ), or 40 mg/ml (F). The line above is the calculated amount of radioactivity in each well (2.1 ⫻ 10 6 dpm at 200 ␮M ATP), based on the specific gravity of the radiolabeled ATP on the day of the experiment. Data are plotted on a log–log scale.

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FIG. 6. Determination of K i(ATP-␥-S) for HPV6 E1. As described under Materials and Methods, 120-min timecourses were run using 10 nM HPV6 E1 with 7.5– 60 ␮M ATP-Mg plus 500 ␮M excess MgOAc and with 0 –100 ␮M ATP-␥-S. (A) Relationship between concentration of P i product (determined by TLC) and cpm determined by scintillation proximity, for the last timepoint of reactions at four concentrations of ATP (7.5– 60 ␮M) and two concentrations of ATP-␥-S (0 or 25 ␮M); linear regression gave a slope of 0.28 nM P i per cpm. (B) Product versus time data for two of the reactions, 30 ␮M ATP and 0 (}) or 100 (■) ␮M ATP-␥-S. Error bars represent standard deviations for three reactions run in parallel. Slopes of 7.5 ⫻ 10 ⫺10 Ms ⫺1 (r 2 ⫽ 0.9990) and 1.6 ⫻ 10 ⫺10 Ms ⫺1 (r 2 ⫽ 0.997) were determined by linear regression. (C) Observed rate (in pM P i produced per s) for substrate concentrations ranging from 7.5 to 60 ␮M at ATP-␥-S concentrations of 0 (F), 3.1 ␮M (■), 6.3 ␮M (}), 12.5 ␮M (Œ), 25 ␮M (*), 50 ␮M (E), and 100 ␮M (䊐). Lines are derived from the best fit of the data to a model assuming competitive inhibition. (D) Same data plotted in double-reciprocal format.

detected using several different bead concentrations. For each bead concentration, the assay signal was found to be linear over at least three orders of magnitude, up to approximately 100 ␮M P i. It appears that adsorbed and free phosphomolybdate are in equilibrium since, despite the linearity observed at each bead concentration, the signal showed a hyperbolic dependence on bead concentration. Since kinetic experiments are typically run to a maximum of 20% conversion, substrate concentrations of up to at least 500 ␮M could be used without modification of the assay procedure. We have also found that reactions at even higher levels of product could be studied by diluting the quenched reaction mixture prior to the addition of SPA beads. At these higher substrate concentrations, however, the sensitivity of this procedure does not necessarily offer any advantage over colorimetric methods. Even at the highest concentration of beads used, the signal was about one-half of the calculated dpm, based on the calibrated specific activity of the [␥- 33P]ATP (see Fig. 5). Part of this difference is due to the efficiency of the scintillation detection, since similar levels of counts were obtained using scintillation fluid and a standard scintillation counter. To investigate the utility of the SPA procedure for

mechanistic studies, initial velocity kinetics were carried out to determine the mechanism of inhibition and the value of K i for the substrate analog ATP-␥-S. As described under Materials and Methods, aliquots were removed from large reactions at times ranging from 20 to 120 min. To convert the raw cpm data to the molar concentration of P i product, detection at the last timepoint was carried out by both TLC and SPA for several reactions. TLC has the unique advantage that both substrate and product can be measured simultaneously, whereas other methods require comparison to phosphate standards for quantitation. Thus, the molar concentration of P i can be determined from the known initial substrate concentration and the measured proportion of P i product. In Fig. 6A, cpm from SPA detection are plotted vs the product concentration determined for the same reaction timepoint by TLC. The slope of the best line through these points was used as a conversion factor for all the SPA data. The slope varies with each experiment depending on the age of the radioactivity and the specific activity used per well. Time courses were then used to obtain observed velocities at each concentration of inhibitor and substrate. Figure 6B shows representative data, obtained at 30 ␮M ATP and 0 or 100 ␮M ATP-␥-S. Velocities of

SCINTILLATION PROXIMITY ATPase ASSAY

750 and 160 pM/s, respectively, were obtained by linear regression. Note that for the higher inhibitor concentration, the conversion of substrate ranged only from 0.7 to 4%, yet the precision of detection is maintained. The full dataset, with four substrate and six inhibitor concentrations plus controls, consisted of 28 such time courses run in triplicate (Fig. 6C). Points correspond to measured velocities, whereas lines are derived from the best fit of the data to a model assuming competitive inhibition, as determined by nonlinear regression. The same pattern is replotted in doublereciprocal (Lineweaver–Burke) format in Fig. 6D. The values obtained for k cat (7 min ⫺1) and K m (13 ␮M) agree well with those determined for this enzyme in the absence of inhibitor (1). The fitted value of K i was 8 ␮M, and the good fit of the data to the competitive model (␹ 2 ⫽ 250) was in clear contrast to alternative models; for example the best fit to a model assuming noncompetitive inhibition (inhibitor binding independent of substrate binding) gave a ␹ 2 of 1790. We have carried out similar mechanistic studies using only the TLC procedure, but for such a large number of data points this proved to be very laborious. Furthermore, although we have obtained roughly similar kinetic parameters with both methods, we found sample-to-sample and day-to-day variation to be consistently higher for TLC; thus in our hands it has not been possible to make definite mechanistic conclusions based on TLC data alone. DISCUSSION

Although our interest has been the study of ATPases, especially the E1 helicase of papillomaviruses, methods to detect inorganic P i are broadly applicable since this species is a product of many other enzymes, for example GTPases, phosphatases, or inorganic pyrophosphatases. Furthermore, protein kinases and other phosphoryl transfer enzymes may yield free P i in the absence of the second (acceptor) substrate; so phosphate detection methods could be applied to the study of these proteins also. It has been shown, for example, that inhibitors of p38 MAP kinase inhibit its intrinsic ATPase activity with similar potency (24). Many of these phosphate-releasing or phosphoryl-transfer enzymes bind substrates in the low- to submicromolar range; so this sensitive SPA methodology could be particularly useful for their study. The scintillation proximity assay described here should be well suited to automated high-throughput screening since it involves only the sequential addition of reagents; no filtration or transfer steps are needed. Furthermore, the signal is relatively insensitive to small variations in the concentrations or volumes of detection reagents and is stable with respect to counting delays ranging from 1 h to several days. Other high-throughput ATPase assays also rely on the for-

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mation of phosphomolybdate, but use a colorimetric detection procedure. (Previously described assays in which radiolabeled P i is incorporated into phosphomolybdate require a physical separation step and thus are not amenable to high throughput.) The colorimetric procedures using malachite green are very straightforward to perform and have the advantage that they do not require radioactivity. However, they are not sensitive enough to detect low- or subnanomolar concentrations of product and thus are not appropriate methods for some targets. Furthermore, in practice the color change produced by formation of charge-transfer complexes with cationic dyes is much less stable than the scintillation signal generated in our procedure, usually due to slow precipitation of the phosphomolybdate– dye complex. Although several groups (17, 25, 26) have demonstrated that detergents or other additives stabilize the dye–phosphomolybdate signal for up to several hours, in the scintillation proximity procedure no deterioration is observed after 24 h or longer, possibly because adsorption to the bead surface stabilizes the phosphomolybdate. This allows multiple assay plates, generated over a period of time, to be read together without variability due to signal decay. Prolonged signal stability has also proven beneficial when occasional equipment malfunction prevented prompt acquisition of data. Several coupled assays have been reported in which the P i product serves as a substrate for a second enzyme. Although not usually suitable for screening, these methods can be very useful for kinetic studies. For example, in the method of Webb (27), purine nucleoside phosphorylase converts 7-methyl-6-thioguanosine and P i to ribose 1-phosphate and 2-amino6-mercapto-7-methylpurine to give an absorbance increase at 360 nm. This method has the great advantage of a continuous readout, so that it is not necessary to stop reactions prior to detection. However, the lower limit of detection is reported to be 2 ␮M P i in a 1 ml volume. A further limitation for all coupled assays is that one must ensure that the coupling steps are not rate limiting, under all assay conditions studied. The assay of Webb is particularly limited in this regard, since one is restricted to a pH range of 6.5– 8, though modifications have been reported which allow its use over a much broader pH range (28). In contrast, the accuracy of the scintillation proximity method is not affected by the buffer conditions used for the enzymatic reactions, since product is detected in a separate step at low pH. In a more sensitive coupled procedure, the fluorescent molecule resorufin is generated from nonfluorescent Amplex Red, giving rise to a signal which can be detected at concentrations as low as 0.2 ␮M (29). However, this even more complex method requires three coupling enzymes and is not a continuous assay. In summary, we feel that the simple and robust scintillation proximity ATPase method described here

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offers unique advantages over other phosphate detection methods. It is particularly suited to screening applications, since it requires few components and no transfer, separation, or washing steps. It is also well suited to kinetic studies since one can work over a broad substrate concentration range, down to the low nanomolar in principle, and over a very wide range of reaction conditions. ACKNOWLEDGMENTS We thank Dr. Michael August for helpful discussions and Drs. Jacques Archambault, Pierre Bonneau, and Stephen Mason for critical reading of the manuscript.

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