Welcome to www.atpsynthase.info! Updated 04. July 2012
Home Enzyme overview Recent papers Researchers Links Images Protocols
  Search The Web / This site   for  

Lab protocols useful for ATP synthase studies

On this page I discuss some general aspects of methods used in ATP synthase research. I have also published here some of the protocols I had actually used, so you are welcome to ask any questions on them if something goes wrong or if something is unclear.

Table of Content

ATP synthesis measurements
     General considerations
     Measurement of ATP synthesis in chromatophores from Rhodobacter capsulatus.
     Measurement of ATP synthesis in Escherichia coli inverted membrane vesicles (luciferase).
     Measurement of ATP synthesis in Escherichia coli inverted membrane vesicles (pH assay).
ATP hydrolysis (ATPase) activity measurements
     General considerations.
     Measurement of ATP hydrolysis by pH assay
     Measurement of ATP hydrolysis via NADH absorption in ATP regenerating system
     Measurement of ATP hydrolysis by Le Bel (Lebel) method

ATP synthesis measurements

General considerations

Several conditions should be met for precise quantitative measurement of ATP synthesis. First, the enzyme should be incorporated into a membrane. Second, this membrane should be energized (i.e. there should be a sufficiently high  Delta mu H+ present). Third, there should be a way to monitor and to control  Delta mu H+ magnitude. Finally, there should be some way to measure ATP production itself.

In respect to the first condition I would distinguish 3 discrete experimental systems: native photosynthetic membranes, native non-photosynthetic membranes, and artificial membranes.

The easiest experimental system for ATP synthesis measurements are native photosynthetic membranes (derived from plant chloroplasts or phototrophic bacteria): they let you control  Delta mu H+ by simply turning on and off the light. Moreover, the  Delta mu H+ value at saturating illumination is usualy very high in photosynthetic membranes. So by adjusting the power of illumination one can finely and precisely adjust  Delta mu H+ . Moreover, in many cases the intrinsic carotenoid pigments present in native photosynthetic membranes serve as a voltmeter: their absorption spectra shift in response to electric field across the membrane. So if no Delta_pH is present,  Delta mu H+ is composed of transmembrane membrane voltage only and can be directly monitored optically.

Native non-photosynthetic membranes (e.g. submitochondrial particles or inverted bacterial membranes) are a bit less convenient - to turn on  Delta mu H+ it is necessary to add respiration substrates. This limit the time resolution of the measurement, unless a stopped-flow apparatus or caged substrates are used. Another problem is to switch  Delta mu H+ off without uncoupling the membrane: specific inhibitors of the respiratory chain are necessary for that (in contrast to the photosynthetic membranes, where you can just switch off the light). Finally, some method for  Delta mu H+ measurement is necessary if quantitative analysis is required.
Despite these drawbacks, it is relatively easy to measure ATP synthesis in native non-photosynthetic membranes. High  Delta mu H+ magnitude sufficient to drive synthesis reaction can readily be generated. Moreover, sometimes it is possible to use non-respiratory intrinsic enzymes for generation of  Delta mu H+ , such as pyrophosphotase or transhydrogenase.

Artificial membranes (mainly liposomes) have many important advantages over native membranes. The most important advantage is that proteoliposomes are a clearly defined and controlled system. Protein and lipid composition can be precisely manipulated and reproduced between different experiments. One can be sure that there are no "unknown" components unavoidable in native membranes.
Surely, this comes at a price. First, it is necessary to purify ATP synthase and incorporate it into liposomes with minimal loss in activity. This is a very challenging task, and it took years in the few labs that demonstrated high rates of ATP synthesis in proteoliposomes. However, getting proteoliposomes with some ATP synthesis activity is relatively easy.
Second, it is necessary to generate  Delta mu H+ . With liposomes one way is to co-incorporate ATP synthase together with some other  Delta mu H+ generating protein. Bacteriorhodopsin is probably the most popular candidate for this, but any other H+-pumping enzyme is also OK. Of course, co-incorporation makes the sample preparation even more challenging. Another way is to use diffusion potentials and artificial pH gradients (so-called "acid-base transition" method). In this case concentrated liposomes incubated in an acidic buffer (pH about 4-5) with low concentration of K+ are quickly diluted by a base buffer (pH 8-9) that has high K+ concentration and contains K+-ionophore valinomycin. After the dilution positively charged K+ are transported by valinomycin across the membrane into liposomes and charge the membrane (positive inside). A a result, voltage of up to 120mV can be generated on the membrane. Together with Delta_pH (acid inside, alkaline outside), this voltage builds a substantial  Delta mu H+ sufficient for ATP synthesis.
Such approach allows to precisely set the desired  Delta mu H+ and its electric or chemical component. However, this  Delta mu H+ dissipates within tens of seconds, so no steady-state measurements are possible. Another alarming observation is that for ATP synthase such  Delta mu H+ seems to be not equivalent to  Delta mu H+ of the same magnitude generated by proton-pumping proteins. Many experimental observations from different research groups indicate that for the same ATP synthesis rate significantly higher  Delta mu H+ is necessary in acid-base transition than in native membranes.

As for the ATP synthesis detection method, the most widely used approach is luciferin/luciferase chemiluminescence measurement. The advantages of this method are 1) incredibly high sensitivity - ATP concentrations down to femtomolar can be readily and reliably detected; 2) linearity of luciferase response in fM-uM ATP concentration range; 3) ease of use - it is possible to monitor ATP synthesis in real time with 100ms resolution in time. Among the drawbacks are low upper limit of ATP concentration that can be quantitatively measured (<10uM) and sensitivity of luciferase to many factors such as pH, temperature, ion composition, etc.
Among the alternatives to luciferin/luciferase for ATP synthesis detection are measurements of radioactive phosphate incorporation, of pH change accompanying the ATP synthesis at pH>7.2, of NADP+ reduction in enzymatic system containing hexokinase and glucose-6-phosphate dehydrogenase, and some other methods.

Measurement of ATP synthesis in chromatophores from Rhodobacter capsulatus.

Summary
Chromatophores are inverted plasma membrane vesicles. When Rb. capsulatus is grown photothropically, chromatophore membranes contain photosynthetic proteins that can generate high  Delta mu H+ in response to illumination.
Red light (wavelenght > 650nm) is used for excitation; ATP production is monitored via luciferase chemiluminiscence at 560nm. The method works up to 5-10 uM ATP concetration. Temperature should not exceed 40C, pH should be in 6.5-8.5 interval.

Protocol
Chromatophores were suspended to bacteriochlorophill concentration of ~10uM in buffer containing:
100 mM potassium acetate (luciferase activity is suppressed by Cl-, so acetates are used instead of chlorides), 20 mM Glycylglycine, 20 mM Na2HPO4, 5 mM magnesium acetate, 2 mM KCN (to inhibit cytochrome oxydase so that no Delta-muH+ is generated in the dark as that oxygen is not consumed in the cuvette), 2 mM K4[Fe(CN)6] (as an electron donor for photosynthetic reaction centres), 5 uM 1,1'-dimethylferrocene (as redox mediator to deliver electrons from K4[Fe(CN)6] to reaction centres), 30 - 100 uM ADP, 0.2 mM luciferin and 10 U/ml luciferase; pH 8.0.
During steady-state ATP synthesis measurement actinic illumination was provided via a lightguide by a slide projector with a red optical filter (OG590, Schott, Mainz) installed. Luciferase chemiluminescence was detected by Thorn EMI 9256B photomultiplier (UK) shielded against actinic light by a stack of 3 blue filters (BG 39 Schott, Mainz, Germany). Measurements were done at room temperature.
The luciferin-luciferase system was calibrated in each sample by addition of freshly prepared ATP solution. The calibration was linear in the range of 0 to 5 uM final ATP concentration. Slight decrease in the sensitivity (which became more pronounced at higher ATP concentration) during the measurements was taken into account by repetitive calibrations during and after each experiment. In the presence of ADP (without any ATP added) a minor ATP synthesis (up to 10 fM/s per mM BChl) insensitive to ATP syntase inhibitors was observed, probably due to adenylate kinase activity of chromatophores.

Comments

  • Depending on the sensitivity reqirements luciferase and luciferin concentrations can be altered significantly. The higher the luciferase concentration - the higher the sensitivity.
  • The pH optimum of luciferase reaction is about 7.7. However, it is possible to measure from pH 6.5 to 8.5 (but it might be necessary to increase the luciferase concentration)
  • Luciferase reaction with ATP and luciferin requires oxygen. If oxygen consumption is significant, the luciferase response may alter during the experiment, up to complete loss of chemiluminiscence.
  • Other redox-buffers (e.g. ascorbate) can be used instead of K4[Fe(CN)6, and other redox mediators can substitute for 1,1Edimethylferrocene.
  • Commercialy available ADP always have some fraction of ATP present. This ATP leads to a non-zero background chemiluminescence. To make things even worse, there is some adenylate kinase activity in chromatophores, so ATP is slowly formed from ADP. The first problem can be solved by additional purification of ADP (e.g. by incubation with glucose and Mg2+ in the presence of hexokinase and subsequent gel-filtration to get rid of hexokinase). The second problem can be solved by addition of adenylate kinase inhibitor, P1,P5-Di(Adenosine-5')Pentaphosphate(Ap5A).

References

  • Lundin, A., Thore, A., and Baltscheffsky, M.
    Sensitive measurement of flash induced photophosphorilation in bacterial chromatophores by firefly luciferase,
    FEBS Lett. 79 (1977) 73-76.
  • Feniouk, B. A., Rebecchi, A., Giovannini, D., Anefors, S., Mulkidjanian, A. Y., Junge, W., Turina, P., and Melandri, B. A.
    Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force,
    Biochim.Biophys.Acta 1767 (2007) 1319-1330.

Measurement of ATP synthesis in Escherichia coli inverted membrane vesicles (luciferase).

Summary
This method works for E. coli ATP synthase and for recombinant Bacillus PS3 thermophilic enzyme expressed in E. coli strains lacking its own ATP synthase. I do not see any reason why it would not work for other recombinant ATP synthases.
 Delta mu H+ is generated by respiratory chain enzymes: NADH or succinate are good substrates for that. ATP synthesis can be detected by luciferin-luciferase.

Protocol
E. coli inverted membranes (subbacterial vesicles) were diluted to final protein concentration of ~10-100 mkg/ml in medium containing:
100 mM potassium acetate (luciferase activity is suppressed by Cl-, so acetates are used instead of chlorides), 20 mM HEPES, 2-20 mM Na2HPO4, 5 mM magnesium acetate, 50-500 uM ADP, and 2 uM P1,P5-Di(Adenosine-5')Pentaphosphate(Ap5A); pH was 7.9.

Each 2ml sample was prepared by mixing 1.9 ml of the above solution with 100ul of luciferase reagent from Roche's
ATP Bioluminescence Assay Kit CLS II (PDF file, 122kb). Then the sample is placed into an appropriate higly sensitive light detection device, where additions to the sample can be made during the measurement. I have used a Jasco FP-6500 fluorometer with excitation lamp turned off and with 750V voltage on PMT detector.
The reaction was started by addition of NADH (to final concentration of 2 mM) or succinate (to final concentation of 3-5 mM). If desired, the reaction can be stopped by addition of uncoupler (I have used mixture of nigericin+valinomycin added to final concentration of 500nM each)
Calibration was performed in each sample by ATP additions (1 addition before the reaction start, and 2-4 additions after).

Comments

  • If a standard rectangle 1cm cuvette is used for measurement, I recommend to attach a piece of aluminium foil (or some other highly reflective material) to the side of the cuvette that is opposite to the detector. This increases signal-to-noise ratio, because more light emitted by luciferase is reflected into detector. Another option is to buy a special cuvette with mirror coating on one outer wall (here is one example from Hellma GmbH & Co. KG).
  • Although I have used fluorometer for this measurement, it is perfectly all right (and might be advantageous) to use a luminometer that has a built-in sample mixing device and allows to make additions to the sample during the measurement.
  • It should be kept in mind that luciferase chemiluminescence requires oxygen. So if the respiration rate in the sample is high (e.g. very high concentration of membranes used, or uncouplers added), at some moment all the oxygen might be consumed. This will result in sudden decrease of chemiluminescence to zero level. Reducing membranes concentration is possible solution for that problem.
  • As mentioned before, commercial ADP is always contaminated with some traces of ATP. So if ADP is added to the sample after measurement start, a clear stepwise increase in chemiluminescence is observed. After calibration of this stepwise increase with a standart ATP solution, ADP addition can be used as a first calibration, so that no ATP addition is required before reaction start.
    If ADP has too much ATP contamination (i.e. final ATP in the sample exceeds 500nM), additional purification of ADP (e.g. by incubation with glucose and Mg2+ in the presence of hexokinase and subsequent gel-filtration to get rid of hexokinase) might be considered.

References

  • Suzuki, T., Ozaki, Y., Sone, N., Feniouk, B. A., and Yoshida, M.
    The product of uncI gene in F1Fo-ATP synthase operon plays a chaperone-like role to assist c-ring assembly,
    Proc.Natl.Acad.Sci.U.S.A 104 (2007) 20776-20781.

Measurement of ATP synthesis in Escherichia coli inverted membrane vesicles (pH assay).

Summary
This method works for any ATP synthase. The limitations are:
1) generation of  Delta mu H+ must not affect the pH of the bulk phase,
2) the pH of the bulk phase should be higher than 7.2, and
3) the pH buffer capacity should be low.
If these conditions are met, ATP syntesis leads to alkalinization of the bulk phase, which can be registered by pH electrode or by pH-indicator dye.

Protocol
E. coli inverted membranes (subbacterial vesicles) were diluted to final protein concentration of ~10-20 mkg/ml in buffer containing:
100 mM KCl, 2.5 mM MgCl2, 2.5 mM HEPES, 10% glycerol, 2.5 mM K2HPO4, 650 uM ADP, and 50 uM pH-indicator dye Phenol Red; pH was adjusted to 8.0 by KOH.

The reaction was started by addition of potassium succinate to final concentration of 3.3 mM and stopped by addition of either malonate to 10 mM or nigericin/valinomycin to 500nM each. It should be noted that NADH should not be used as a respiration substrate, because its oxidation itself causes pH change in the sample. The pH changes accompanying ATP synthesis were measured via absorption changes of Phenol Red at 557 nm in Jasco V550 spectrophotometer. All stock solutions used for additions were titrated to pH 8.0 to avoid pH changes in the sample.
Calibration was performed in each sample by standard H2SO4 or NaOH solutions.

Comments

  • It is stated that the method works above pH 7.2. This limitation comes from pK2 of inorganic phosphate, and the closer to 7.2 is the pH in the sample, the smaller is the pH change caused by ATP synthesis/hydrolysis (see the corresponding FAQ section if you are inerested, why is it so). The optimal pH for this method is about 8.
  • The lower the buffer capacity of the medium, the larger is the pH change caused by ATP synthesis/hydrolysis. However, when the buffer capacity is too low, gradual acidification of the medium by CO2 from air can cause considerable baseline drift. Another problem is the large pH jumps caused by additions of reagents stock solutions that have pH slightly different from the medium.
    So as a rule of thumb, 2-5mM buffer with pK around 7.7-8.0 works best. But if you are aimed at maximal sensitivity and the timescale of the measurement is small, omitting all pH buffers might be an option.
  • When working with pH indicator dye and membranes, one faces the problem of high noise due to scattering. Aggregation of membranes into visible "flakes" that cause huge erratic jumps in sample absorption is also a common problem. The best way around it is to use a double beam spectrophotometer that can simultaneously measure at two wavelengths. In case of phenol red the choice is 557nm and 618nm. Subtracting the trace recorded at 618nm from the 557nm, it is possible to get rid of artefacts caused by scattering and by other absorption changes unrelated to pH change of the sample.

References

  • Nishimura, M., Ito, T., and Chance, B.
    Studies on bacterial photophosphorylation. III. A sensitive and rapid method of determination of photophosphorylation.
    Biochim.Biophys.Acta 59 (1962) 177-182.
  • Feniouk, B. A., Suzuki, T., and Yoshida, M.
    Regulatory interplay between proton motive force, ADP, phosphate, and subunit epsilon in bacterial ATP synthase.
    J.Biol Chem 282 (2007) 764-772.

ATP hydrolysis (ATPase) activity measurements

General considerations.

There are several methods for ATPase activity measurements. One of the oldest experimental approach is to take aliquots from the sample after reaction start and measure the amount of inorganic phosphate generated from ATP hydrolysis. With this apporach there are almost no restrictions on experimental conditions (ion strength, pH, inhibitors, reaction temperature, etc). The obvious limitations of this method are 1) that it cannot be applied in the presence of phosphate, and 2) that ATP and ADP concentrations reciprocally change during the reaction course.
Another approach free of both limitations is to use enzymatic ATP regenerating system that couples ATP hydrolysis to NADH oxidation and regenerates ATP from ADP. With this approach ATP concentration remains constant, ADP concentration remains negligibly low, and phosphate in high concentration does not interfere with the detection. However, with this method it is impossible to measure ATPase activity in the presence of ADP.
A very affordable and potent method is to monitor the ATPase reaction via acidification of the medium (already described above as a method for ATP synthesis measurement - it obviously works fine both for ATP synthesis and hydrolysis reaction). Below is a detailed protocol for this method.

Measurement of ATP hydrolysis by pH assay

Summary
The equation of ATP hydrolysis at pH > 7.2 (i.e. above the pK2 of inorganic phosphate) is:

ATP4- + H2O <=> ADP3- + Pi2- + H+

This implies that hydrolysis of each ATP molecule produces one H+, so ATPase activity can be followed by change in pH. Technically this can be done either with a sensitive and fast pH electrode or with appropriate pH indicator dyes. An important condition necessary for high sensitivity of this method is low pH-buffering capacity of the sample medium (no more than 1-5mM of pH-buffer should be present).

Protocol
Purified F1, FOF1 or inverted membranes (subbacterial vesicles) were diluted to concentration corresponding to ATP hydrolysis rate of 5-100 uM/min in buffer containing:
100 mM KCl, 2.5 mM MgCl2, 2.5 mM HEPES, 10% glycerol, and pH-indicator dye Phenol Red to absorption of ~1.0 AU at 557nm (at pH 8.0 it was ~50uM); pH was adjusted to 8.0 by KOH.

The reaction was started by addition of ATP to final concentration of 1 mM and stopped by addition of NaN3. In case of membranes, uncoupling effect was assessed by addition of nigericin/valinomycin to 500nM each. ADP, phosphate or other compounds of interest were added during the reaction course. In case the addition noticably changed the buffer capacity of the sample, calibrations before and after the addition were made.
The pH changes accompanying ATP hydrolysis were measured via absorption changes of Phenol Red at 557 nm in Jasco V550 spectrophotometer. All stock solutions used for additions were titrated to pH 8.0 to avoid pH changes in the sample.
Calibration was performed in each sample by standard H2SO4 or NaOH solutions. The H+/ATP ratio under the experimental conditions used was ~0.85.

Comments

  • It is stated that the method works above pH 7.2. This limitation comes from pK2 of inorganic phosphate, and the closer to 7.2 is the pH in the sample, the smaller is the pH change caused by ATP synthesis/hydrolysis (see the corresponding FAQ section if you are inerested, why is it so). The optimal pH for this method is about 8.
  • The lower the buffer capacity of the medium, the larger is the pH change caused by ATP synthesis/hydrolysis. However, when the buffer capacity is too low, gradual acidification of the medium by CO2 from air can cause considerable baseline drift. Another problem is the large pH jumps caused by additions of reagents stock solutions that have pH slightly different from the medium.
    So as a rule of thumb, 2-5mM buffer with pK around 7.7-8.0 works best. But if you are aimed at maximal sensitivity and the timescale of the measurement is small, omitting all pH buffers might be an option.
  • When working with pH indicator dye and membranes, one faces the problem of high noise due to scattering. Aggregation of membranes into visible "flakes" that cause huge erratic jumps in sample absorption is also a common problem. The best way around it is to use a double beam spectrophotometer that can simultaneously measure at two wavelengths. In case of phenol red the choice is 557nm and 618nm. Subtracting the trace recorded at 618nm from the 557nm, it is possible to get rid of artefacts caused by scattering and by other absorption changes unrelated to pH change of the sample.

References

  • Nishimura, M., Ito, T., and Chance, B.
    Studies on bacterial photophosphorylation. III. A sensitive and rapid method of determination of photophosphorylation.
    Biochim.Biophys.Acta 59 (1962) 177-182.
  • Feniouk, B. A., Suzuki, T., and Yoshida, M.
    Regulatory interplay between proton motive force, ADP, phosphate, and subunit epsilon in bacterial ATP synthase.
    J.Biol Chem 282 (2007) 764-772.

Measurement of ATP hydrolysis via NADH absorption in ATP regenerating system

Summary
In this method ATP hydrolysis rate is measured by monitoring ADP production via two enzymatically catalysed reactions. As a result of these two reactions, ADP is converted back to ATP (i.e. regenerated, hence the name of the method).
The essetial components of the ATP regenerating system are (PEP)) and pyruvate kinase. The latter enzyme catalyses phosphorylation of ADP by PEP:

ADP + phosphoenolpyruvate <=> ATP + pyruvate

In turn, pyruvate production is detected via oxidation of NADH by pyruvate catalysed by lactate dehydrogenase:

pyruvate + NADH <=> lactate + NAD+

Oxidation of NADH to NAD+ can be followed opticaly via decrease in NADH absorption at 340nm. So the overall detection sequence is like:

(ATP => ADP) ~ (PEP =>pyruvate) ~ (pyruvate => lactate) ~ (NADH => NAD+) ~ A340

This is a widely used method that have several important advantages. First, ATP concentration is constant during the reaction course (excess PEP should be present in the sample!). Second, it is a real time measurement, so changes in the rate of ATP hydrolysis can be easily studied. This is especially important for ATP synthase that has a complex regulation, so that the initial and the steady-state hydrolysis rates may differ significantly.
The limitations of the method are: 1) no ADP is present in the sample, in contrast to the physiological conditions when both ATP and ADP are present; 2) presence of two extra enzymes (pyruvate kinase and lactate dehydrogenase) limit the experimental conditions; 3) when used for biological membrane preparations, respiratory chain enzymes that oxidize NADH might interfere with the detection.

Protocol
The protocol below is for E. coli membranes, but can be applied to any ATP synthase or F1-ATPase preparation.
E. coli membranes are diluted to protein concentration of 0.01-0.02mg/ml in a buffer containing 20mM HEPES, 5mM MgCl2, 100mM KCl, 5mM KCN, 2.5mM phosphoenolpyruvate, 200uM NADH, 0.1mg/ml pyruvate kinase, 0.1mg/ml lactate dehydrogenase, pH 7.5-8.0.
Reaction is started by addition of ATP to desired final concentration (typically 1 mM) and followed by the decrease in NADH absorption at 340nm wavelength. The molar extinction coefficient of NADH at 340nm is 6220M-1.
Other additions (e.g. inhibitors, uncouplers, LDAO, etc.) can be made during reaction course.
When NADH is exausted (A340 drops below 0.1), another addition of NADH to ~200uM final concentration can be made to continue the measurement.

Comments

  • A good idea is to always check the method with a standard ADP solution: simply add ADP to final concentration of 50-100uM and see if the addition causes a sharp stepwise drop in A340. If such ADP addition causes a gradual smooth decrease in A340, the reaction rate is limited by insufficient activity of either pyruvate kinase or lactate dehydrogenase. Increasing the enzyme concentration or using a fresh stock should solve the problem.
  • In the protocol above potassium cyanide is added to inhibit NADH oxydation by E.coli respiratory chain. However, in reality KCN does not fully block NADH oxidation by membranes. So the baseline before ATP addition has some slope that should be taken into account during ATP hydrolysis rate calculations.
    Another option to block NADH oxydation unrelated to ATPase reaction when working with native membranes is to use piericidin (~2nmol/mg membrane protein) - a specific inhibitor of NADH:ubiquinone oxidoreductase.
  • The method can also be used as a control for other ATP hydrolysis assays - one can easily measure the final ADP concentration in a sample.

References

  • Pullman,M.E., Penefsky,H.S., Datta,A. and Racker,E.
    Partial resolution of the enzyme catalyzing oxidative phosphorylation. I. Purification and properties of soluble, dinitrophenol-stimulated adenosine triphosphatase.
    J.Biol.Chem. 235 (1960) 3322-3329.
  • Feniouk, B. A., Suzuki, T., and Yoshida, M.
    Regulatory interplay between proton motive force, ADP, phosphate, and subunit epsilon in bacterial ATP synthase.
    J.Biol. Chem. 282 (2007) 764-772.

Measurement of ATP hydrolysis by Le Bel (Lebel) method

Summary
In this method ATP hydrolysis rate is measured by monitoring phosphate production. This is an end-point assay, so the ATP hydrolysis reaction must be stopped first (e.g. by
TCA addition), and than the phosphate concentration can be determined.

Protocol
ATP hydrolysis reaction is started either by addition of ATP or of the enzyme. At a given time an aliquot of 0.5ml reaction mixture is taken and thoroughly mixed with 0.5ml ice-cold 0.5 N TCA. Then 0.5ml freshly made Le Bel Reagent (3.6 M acetic acid, 0.66 M sodium acetate, 20 mM copper sulfate; made from stock solutions of LeBel components A:B:C in proportion 6:1:1) is added, and the sample is mixed thoroughly. Then the sample is incubated for 5 minutes. After that the sample is filtered or centrifuged to remove insoluble material, and the absorption of the resultant clear solution is measured at wavelength of 745 nm.
The method works well for phosphate concentration in the range of 0 to 400 uM (final value in the sample). The calibration curve should be made accordingly, by adding known phosphate concentration and stopping the reaction immediately after start.

Comments

  • This is a very robust method that can be used in almost any experimental conditions (but phosphate should be excluded from the reaction medium, of course!). Care should be taken during calibration to ensure that all factors present in the sample measurement (membranes, detergents, etc.) are also present during calibration.
  • As an end-point assay, this method cannot be used to determine the initial rate of reaction. However, it is possible to follow slow (tens of seconds scale) changes by taking aliquotes from the reaction mixture at short time intervals.
  • A modification of this method was developed by Hicks and Krulwich (see Ref. 2 below), where the detection solution is a 4:5:5-mixture of LeBel reagent (3.6 M acetic acid, 0.66 M sodium acetate, 20 mM copper sulfate) : (5% solution of ammonium molybdate) : (5% sodium sulfite + 2% p-methylaminophenyl sulfate solution in proportion).

References

  • LeBel, D., G. G. Poirier, and A. R. Beaudoin.
    A convenient method for the ATPase assay.
    Anal. Biochem. 85 (1978) 86-89.
  • Hicks, D. B., and T. A. Krulwich.
    Purification and reconstitution of the F1FO-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+. J. Biol. Chem. 265 (1978) 20547-20554.




All rights reserved. Copyright Boris A. Feniouk, 2002-2012
On the COPYRIGHT STATUS and TERMS OF USE of images or text from www.atpsynthase.info