8
Sensing of adenosine-5 0 -triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye Dmytro A. Yushchenko a,b , Olga B. Vadzyuk c , Sergiy O. Kosterin c , Guy Duportail b, * , Yves Me ´ly b , Vasyl G. Pivovarenko a, * a Department of Chemistry, Kyiv National Taras Shevchenko University, 01033 Kyiv, Ukraine b Photophysique des Interactions Biomole ´culaires, UMR 7175-LC1 du CNRS, Institut Gilbert Laustriat, Faculte ´ de Pharmacie, Universite ´ Louis Pasteur, 67401 Illkirch, France c O.V. Palladin Institute of Biochemistry, 01030 Kyiv, Ukraine Received 12 April 2007 Available online 8 May 2007 Abstract The current work demonstrates the formation of complexes between the tetraanion adenosine-5 0 -triphosphate (ATP) and the flavone derivative 3-hydroxy-4 0 -(dimethylamino)flavone (FME). Two kinds of complexes are evidenced. The higher affinity ATP–FME complex corresponds to a stacking of the two aromatic molecules and leads to a strong hypochromicity of the absorption spectrum of the dye. The lower affinity (ATP) 2 –FME complex results in a strong increase of the fluorescence intensity (20-fold), due mainly to the appearance of the anionic form of FME, as shown by the important red shift (60 nm) of both excitation and emission spectra. Molecular modeling indicates that this anionic form results from the deprotonation induced by the influence of the tetra-charged triphosphate group of the ATP molecules. Using its strong enhancement of fluorescence intensity in the presence of ATP, the dye was used successfully to monitor the succinate-induced production of endogenous ATP in mitochondria. As a consequence, FME can be considered as a starting point to design efficient ATP sensors. Ó 2007 Elsevier Inc. All rights reserved. Keywords: ATP sensing; 3-Hydroxyflavone dyes; Fluorescent probe; Mitochondria Despite the key role of adenosine-5 0 -triphosphate (ATP) 1 , no accurate method of determination of its local concentration in living cells is actually available. The deter- mination of local ATP concentration and its changes with time in live cells is a complex problem not only due to the restricted space and/or time scale limitations but also due to the selectivity required in the determination of this pecu- liar anion. Indeed, a large cohort of anions with close struc- ture and properties exists in cells, including adenosine mono- di-, and triphosphates as well as guanosine, inosine, uridine, and cytidine corresponding anions, nicotinamide adenine dinucleotide (b-NAD), and anions such as phos- phate and pyrophosphate. All of these compounds are putative competitors for any analytical method aimed to detect ATP. To unravel such a complicated task, fluorescence microscopy and fluorescent probes were shown to be effec- tive for measuring the local concentrations of a large num- ber of compounds [1]. For instance, probes were designed to determine nanomolar Ca 2+ concentrations in the presence of 10 4 to 10 5 molar excess of other cations such as Mg 2+ , Na + , and K + [2]. Several attempts were also 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.05.005 * Corresponding authors. Fax: +33 90244313 (G. Duportail); fax: +380 442898391 (V.G. Pivovarenko). E-mail addresses: [email protected] (G. Duportail), [email protected] (V.G. Pivovarenko). 1 Abbreviations used: ATP, adenosine-5 0 -triphosphate; b-NAD, nicotin- amide adenine dinucleotide; GTP, guanosine triphosphate; 3HF, 3-hydroxyflavone; FME, 3-hydroxy-4 0 -(dimethylamino)flavone; F3ME, 3-methoxy-4 0 -(dimethylamino)flavone; Hepes, 4(2-hydroxyethyl)-1-pipe- razineethansulfonic acid; Tris, tris-(hydroxymethylamino)-methane; EDTA, ethylenediaminetetraacetic acid. www.elsevier.com/locate/yabio Analytical Biochemistry 369 (2007) 218–225 ANALYTICAL BIOCHEMISTRY

Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

Embed Size (px)

Citation preview

Page 1: Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

www.elsevier.com/locate/yabio

Analytical Biochemistry 369 (2007) 218–225

ANALYTICAL

BIOCHEMISTRY

Sensing of adenosine-5 0-triphosphate anion in aqueous solutionsand mitochondria by a fluorescent 3-hydroxyflavone dye

Dmytro A. Yushchenko a,b, Olga B. Vadzyuk c, Sergiy O. Kosterin c, Guy Duportail b,*,Yves Mely b, Vasyl G. Pivovarenko a,*

a Department of Chemistry, Kyiv National Taras Shevchenko University, 01033 Kyiv, Ukraineb Photophysique des Interactions Biomoleculaires, UMR 7175-LC1 du CNRS, Institut Gilbert Laustriat, Faculte de Pharmacie,

Universite Louis Pasteur, 67401 Illkirch, Francec O.V. Palladin Institute of Biochemistry, 01030 Kyiv, Ukraine

Received 12 April 2007Available online 8 May 2007

Abstract

The current work demonstrates the formation of complexes between the tetraanion adenosine-5 0-triphosphate (ATP) and the flavonederivative 3-hydroxy-4 0-(dimethylamino)flavone (FME). Two kinds of complexes are evidenced. The higher affinity ATP–FME complexcorresponds to a stacking of the two aromatic molecules and leads to a strong hypochromicity of the absorption spectrum of the dye. Thelower affinity (ATP)2–FME complex results in a strong increase of the fluorescence intensity (�20-fold), due mainly to the appearance ofthe anionic form of FME, as shown by the important red shift (60 nm) of both excitation and emission spectra. Molecular modelingindicates that this anionic form results from the deprotonation induced by the influence of the tetra-charged triphosphate group ofthe ATP molecules. Using its strong enhancement of fluorescence intensity in the presence of ATP, the dye was used successfully tomonitor the succinate-induced production of endogenous ATP in mitochondria. As a consequence, FME can be considered as a startingpoint to design efficient ATP sensors.� 2007 Elsevier Inc. All rights reserved.

Keywords: ATP sensing; 3-Hydroxyflavone dyes; Fluorescent probe; Mitochondria

Despite the key role of adenosine-5 0-triphosphate(ATP)1, no accurate method of determination of its localconcentration in living cells is actually available. The deter-mination of local ATP concentration and its changes withtime in live cells is a complex problem not only due to therestricted space and/or time scale limitations but also due

0003-2697/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2007.05.005

* Corresponding authors. Fax: +33 90244313 (G. Duportail); fax: +380442898391 (V.G. Pivovarenko).

E-mail addresses: [email protected] (G. Duportail),[email protected] (V.G. Pivovarenko).

1 Abbreviations used: ATP, adenosine-50-triphosphate; b-NAD, nicotin-amide adenine dinucleotide; GTP, guanosine triphosphate; 3HF,3-hydroxyflavone; FME, 3-hydroxy-40-(dimethylamino)flavone; F3ME,3-methoxy-40-(dimethylamino)flavone; Hepes, 4(2-hydroxyethyl)-1-pipe-razineethansulfonic acid; Tris, tris-(hydroxymethylamino)-methane;EDTA, ethylenediaminetetraacetic acid.

to the selectivity required in the determination of this pecu-liar anion. Indeed, a large cohort of anions with close struc-ture and properties exists in cells, including adenosinemono- di-, and triphosphates as well as guanosine, inosine,uridine, and cytidine corresponding anions, nicotinamideadenine dinucleotide (b-NAD), and anions such as phos-phate and pyrophosphate. All of these compounds areputative competitors for any analytical method aimed todetect ATP.

To unravel such a complicated task, fluorescencemicroscopy and fluorescent probes were shown to be effec-tive for measuring the local concentrations of a large num-ber of compounds [1]. For instance, probes were designedto determine nanomolar Ca2+ concentrations in thepresence of 104 to 105 molar excess of other cations suchas Mg2+, Na+, and K+ [2]. Several attempts were also

Page 2: Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

Sensing of ATP anion / D.A. Yushchenko et al. / Anal. Biochem. 369 (2007) 218–225 219

undertaken to design fluorescent probes for measuringATP concentrations in aqueous solutions. Hosseini [3]and Huston and coworkers [4] were the first to use synthe-sized fluorescent sensors for detection of polyanions, nota-bly ATP tetraanion. Later a series of ATP chemosensorswas developed [5], and recently guanosine triphosphate(GTP) sensors were proposed [6–8]. All of these groupswere following the same strategy, that is, to elaborate poly-cationic aromatic sensors able to bind in aqueous solutionsa number of anionic species such as phosphate, pyrophos-phate, polycarboxylates, and other similar anions. Unfor-tunately, these sensors did not selectively detect ATPanion and were not efficient in the ATP concentrationrange existing in live cells. For this reason, the biolumines-cence luciferase reaction has remained the method usedmost often for the determination of ATP [9–11].

The design of an ideal ATP fluorescence sensor needs totake into account the peculiarities of the structure of theATP molecule. In aqueous solutions at physiological pH,ATP exists as a tetra-charged anion that can be formallydivided into two parts that provide an amphiphilic charac-ter to the molecule. Both phosphate and ribose residuesform the charged hydrophilic part of the molecule, whereasthe uncharged adenine residue forms the planar and morehydrophobic part. Whereas the hydrophilic part ensures agood solubility of ATP in water and generates an electro-static field around it, the hydrophobic part is required forassociation with the planar and hydrophobic moleculesinvolved in the biochemical reactions with ATP.

Thus, by taking into account these specific features of theATP molecule, an ideal fluorescent probe for ATP detectionnot only should associate with it, through electrostatic inter-action with the positively charged residue and through stack-ing interactions with the hydrophobic adenine residue, butalso should be sensitive to the electrostatic field generatedaround the ATP molecule. To our knowledge, none of thepreviously developed ATP probes [3–5] exhibits a sensitivityto this electric field. In this respect, the major issue was toimprove the probe design so as to increase the bindingparameters and the selectivity of the probe response towardthe ATP anion. Due to our intensive development of newmultiparametric probes based on the 3-hydroxyflavone(3HF) family, we attempted to find possible ATP sensors,while satisfying the previous considerations, among thispromising class of fluorescent probes.

Scheme 1. Chemical structures of ATP

Indeed, these 3HF probes present a strong sensitivity toelectric fields in solution as well as in molecular assemblies.This was shown for local fields generated by molecules [12–20] or ions [21–28] in solution, by membrane potentials[29–33], and by the charged poles of an electrooptical cell[34–36]. Moreover, according to quantum chemical calcu-lations, 3HFs can adopt a totally planar conformation thatis favorable for stacking interaction [37,38]. In addition,the subfamily of 4 0-dialkylaminoflavonols is characterizedby a highly polarizable moiety with a positive charge ableto interact with the negative charges of ATP, thereby fixingan appropriate orientation of the probe molecule in theformed complex. Thus, this last family appears to possessall of the prerequisites to bind ATP and monitor this bind-ing by fluorescence. The potentiality of these compounds toselectively detect ATP in aqueous solutions was confirmedin a preliminary work [39]. The aim of the current workwas to characterize the stoichiometry and dissociation con-stants of the complexes between 3-hydroxy-4 0-(dimethyla-mino)flavone (FME) and ATP in the absence andpresence of 250 mM sucrose. This last experimental condi-tion was needed for the experiments to demonstrate thatthis probe is able to sense the endogenous production ofATP in mitochondria induced by the addition of succinateions.

Materials and methods

FME and 3-methoxy-40-(dimethylamino)flavone (F3ME)were prepared according to Ormson and coworkers [40]and purified after additional crystallization from ethanol(Scheme 1). Their purity was checked by thin-layer chro-matography on Silica Gel 60 (F-254, Selecto Scientific) inchloroform–methanol mixtures (98:2, 95:5, and 90:10,v/v). 4(2-Hydroxyethyl)-1-piperazineethansulfonic acid(Hepes), tris-(hydroxymethylamino)-methane (Tris), andnucleoside phosphates were purchased from Sigma–Aldrich, and sucrose (biochemistry grade) was purchasedfrom Merck.

Absorption spectra were recorded on a Cary 4 spectro-photometer (Varian). Fluorescence spectra were recordedon either a FluoroMax 3.0 (HORIBA Jobin Yvon) or aHitachi MPF-4 spectrofluorometer, with the latter beinghomemade computerized. In all cases, the starting concen-tration of the FME solution (1.5 ml) was 2 lM in 15 mM

and of probes FME and F3ME.

Page 3: Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

220 Sensing of ATP anion / D.A. Yushchenko et al. / Anal. Biochem. 369 (2007) 218–225

Tris buffer (pH 7.4). Titration with ATP was performed byadding stepwise aliquots (2–500 ll) of a 10- to 40-mM ATPsolution. Spectra were recorded at 25 �C after eachaddition.

The molecular geometry of the probes was calculated byAM1 or PM3 semiempirical methods [41] using theMOPAC 6.0 program.

On the assumption of a 1:1 complex, the dissociationconstant KD values were calculated according to theFletcher–Pawl algorithm by using the following commonformula [42,43]:

R ¼ Rc½ATP � þ RFMEKD

½ATP � þ KD

;

where RC and RFME are either the absorbance at 400 nm(A400) or the fluorescence intensity at 488 nm (I488) on theexcitation spectra of the ATP–FME complex and freeFME, respectively, and [ATP] is the concentration of freeATP.

Uterus mitochondria were isolated from rats, whichwere estrogenized 24 h before the experiment. Theisolation procedure was performed in ice-cold sucrosebuffer (250 mM sucrose, 10 mM Hepes, 1 mM ethylene-diaminetetraacetic acid [EDTA], pH 7.4) by twosequential centrifugations, first at 1000g and then at12,000g for the supernatant. Pellets were resuspendedin 500 ll EDTA–sucrose buffer. The isolated mitochon-dria were stored on ice and used within 2 h. Fluoromet-ric experiments with mitochondria were performed at37 �C.

Fig. 1. Absorption (A,C) and fluorescence excitation (B,D) spectra of FME padded either in the absence (A,B) or in the presence (C,D) of 250 mM sucros

Results and discussion

Absorption and excitation spectra

Absorption and excitation spectra of FME in the pres-ence of ATP were performed in 15 mM Tris buffer (pH7.4). As shown in Fig. 1A, the progressive addition ofATP to a 2-lM solution of FME induces two subsequentsteps. The first step is characterized by approximately a2-fold decrease of the initial absorbance of FME. In con-trast, the second step ([ATP] > 0.6 mM) shows only a lim-ited decrease of absorbance but shows a strong red shift(bathochromism) of the maximum wavelength of theabsorption spectrum (from 407 to 450 nm). This behaviorsuggests the formation of at least two types of complexesbetween ATP and FME. An isosbestic point is observedin the 0.6- to 2-mM range of ATP concentration, indicatingthe presence of two states in this concentration range. Theabsorption spectra can be compared with the excitationspectra (Fig. 1B) in the same conditions. The addition ofincreasing amounts of ATP also leads to a biphasic behav-ior with two subsequent steps. In a first step ([ATP] <0.3 mM), the fluorescence intensity increases slightly, butthe excitation spectra are progressively shifted to the red,from 420 to 475 nm. In a second step ([ATP] > 0.3 mM),the fluorescence intensity increases up to approximately20-fold, but with a further shift to the red. Thus, the fluo-rescence quantum yield of the final ATP–FME complexappears to be much higher than the low quantum yieldsof the free FME and the first complex.

robe in 15 mM Tris buffer (pH 7.4). Increasing ATP concentrations weree. Excitation spectra were recorded at 555 nm. arb.u., arbitrary units.

Page 4: Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

Sensing of ATP anion / D.A. Yushchenko et al. / Anal. Biochem. 369 (2007) 218–225 221

Next the absorption and excitation spectra wererepeated in the presence of 250 mM sucrose to model theexperimental conditions used to measure the ATP concen-trations of mitochondria (vide infra). The absorption spec-tra (Fig. 1C) show that a high sucrose concentration affectsthe interaction between ATP and FME. Although a similarlevel of hypochromism is obtained, the red shift is limitedand no isosbestic point can be observed in the presenceof sucrose. An increase of the fluorescence intensity is alsoobserved with increasing concentrations of ATP (Fig. 1D)but is only approximately 2-fold as compared with 20-foldin the absence of sucrose.

Nature of complexes

At ATP concentrations less than 0.6 mM, the largedecrease in the absorption spectra clearly indicates thatATP and FME form a 1:1 stacking complex between theirtwo aromatic rings [44]. The absence of shift in absorption,as well as the small changes in fluorescence intensity, indi-cates that the stacking likely occurs with FME in its neutralform. The stacking complex could be stabilized by an elec-trostatic interaction between the electric dipole of FMEand the negative phosphate group. At higher ATP concen-trations (>0.6 mM), the strong shift of the absorption spec-tra and the isosbestic point, together with the strongfluorescence increase, is consistent with the binding of asecond ATP molecule to the ATP–FME complex. Similarstrong shifts of the absorption and excitation spectra wereobserved previously when the neutral form of 3-hydroxyf-lavones was changed to the anionic form [45]. As a conse-quence, the binding of the second ATP probably inducesthe anionic form of FME. To check this hypothesis, theemission spectrum of the (ATP)2–FME complex was com-pared with the emission spectrum of free FME at pH 7.4and pH 11.7 (Fig. 2). In excellent agreement with ourassumption, the normalized emission spectrum of the com-plex fully matches with the emission spectra of the anionicform of FME at high pH. This formation of the anionic

Fig. 2. Normalized emission spectra of FME probe in aqueous buffer(15 mM Tris, 25 �C) in the absence of ATP at pH 7.4 (—) and pH 11.7 ( )and emission spectra in the presence of 7.5 mM ATP at pH 7.4 of FMEprobe (– - - –) and F3ME probe (– – –). The excitation wavelength is430 nm in all cases. arb.u., arbitrary units.

species of FME may be induced by the polarization effectof the four negative phosphate groups of ATP. A similarinduction of the anionic form of FME was observed previ-ously due to the polarization effect of the negativelycharged membrane surface of anionic egg yolk phosphati-dylglycerol vesicles [45]. This ATP-induced anionic formwas further confirmed by substituting FME with F3MEthat cannot give any anionic form due to the substitutionof the hydroxyl group by a methoxy group. As expected,the interaction of ATP with F3ME provided an increaseof its fluorescence intensity but no shift of its emission max-imum (Fig. 2).

The qualitative similarities in the absorption and excita-tion spectra of FME in the presence of sucrose with thecorresponding spectra in the absence of sucrose suggestthat both ATP–FME and (ATP)2–FME in the presenceof sucrose also form. However, the quantitative differencesin the shift of the excitation spectra (35 nm vs. 55 nm in theabsence of sucrose) and the fluorescence intensity increase(2-fold vs. 20-fold) need to be explained. The 8-fold higherfluorescence quantum yield of free FME in the presence ofsucrose as compared with buffer (cf. Fig. 1D and B) sug-gests a direct interaction between sucrose and FME. Themolecules of sucrose likely form a solvation shell aroundthe FME molecule substituting the water molecules at leastpartly and, thus, decreasing the fluorescence quenchingeffect of water [46,47]. Moreover, due to its lower dielectricconstant in comparison with pure water, sucrose likelyallows a larger repulsion between the two highly chargedphosphate groups in the 2:1 complex. Consequently, thephosphate groups will be more distant from the FME moi-ety in sucrose than in water and, thus, induce the anionicform of FME less efficiently. The less efficient formationof this anionic form in the 2:1 complex in sucrose is sub-stantiated by its less red-shifted absorption spectrum(Fig. 1C) as well as by the different shape and position ofits excitation spectrum (Fig. 1D) with respect to those inthe absence of sucrose.

To further characterize the complexes between ATP andFME, the binding constants of the first ATP–FME com-plex and the final (ATP)2–FME complex were determined.Because in both the absence and presence of sucrose theintermediate and final complexes form in well-separatedranges of ATP concentrations, the binding of the twoATP molecules can be considered as sequential. Conse-quently, their dissociation constants can be determinedindependently from the hypochromicity of the absorptionspectra (Kd1) and the increase of the fluorescence intensitydetermined on the excitation spectra (Kd2), respectively(Fig. 3). A difference of approximately two orders of mag-nitude was observed between the binding of the first ATP(Kd1 = 8 ± 1 Æ 10–5 mol L–1) and the second one (Kd2 =7.4 ± 0.3 Æ 10–3 mol L–1) in the absence of sucrose. The lessfavorable binding of the second ATP suggests either thatthe two binding sites are different or that the binding ofthe first ATP hinders the binding of the second one. Inter-estingly, in the presence of sucrose, Kd1 and Kd2 values of

Page 5: Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

Fig. 3. Values of log[ATP] versus either absorbance at 400 nm, corre-sponding to Fig. 1A (D) and C (m), or fluorescence intensity at 488 nm,corresponding to Figs. 1B (s) and D (d). The ATP concentration rangesare the ones used to calculate Kd values: 0 to 0.6 mM (Fig. 1A and C), 0.3to 4.0 mM (Fig. 1B), and 0.3 to 7.5 mM (Fig. 1D).

222 Sensing of ATP anion / D.A. Yushchenko et al. / Anal. Biochem. 369 (2007) 218–225

7 ± 1 Æ 10–5 and 14.1 ± 0.6 Æ 10–3 mol L–1, respectively, wereobtained, indicating that sucrose interferes with the bindingof only the second ATP molecule to FME.

Finally, the possible structures of the ATP–FME com-plexes were simulated by quantum chemical calculationsin vacuo. Fig. 4A and B present the two possible configu-rations of the 1:1 complex, and Fig. 4C presents the possi-ble configuration of the 2:1 complex. In all cases, the firstATP binds in a parallel orientation to the FME aromaticring, at a short distance (�3.6 A), confirming that intermo-lecular stacking interactions play a major role in the forma-tion of the 1:1 complex. The orientations of the FME andATP molecules in the 1:1 complex reveal that the positivepart of the molecular dipole of FME is close to the anionic

Fig. 4. Possible structures of ATP–FME (A,B) and (ATP)2–FME (C)complexes obtained by quantum chemical calculations in vacuo. Carbonand phosphorus atoms are in dark gray, oxygen and nitrogen atoms are inlight gray, and hydrogen (shown only in right drawings) are in white.

phosphate group of ATP and, thus, stabilizes the complex.In the 2:1 complex, the second ATP molecule binds in thesame orientation as the first one but on the opposite side ofthe FME molecule. In this complex, the close proximity ofthe two negatively charged tetraanions probably is allowedby the positively charged part of the FME molecule that issandwiched between them. These negatively charged tet-raanions strongly polarize the FME chromophore and,thus, favor an intermolecular proton transfer from thehydroxyl group of FME to one of the ATP molecules.Because the resulting anionic form of FME is producedonly in the presence of ATP and not with other nucleotidetriphosphates, this suggests that the adenine base plays therole of proton acceptor in the intermolecular proton trans-fer. A similar intermolecular proton transfer with FMEanion formation was observed when FME interacts withphosphatidylglycerol in Hepes buffer [45]. Finally, ourinterpretation is further substantiated by the negative influ-ence of sucrose on the anion formation. Indeed, by decreas-ing the dielectric constant of the medium, the sucrosemolecules allow an increased repulsion between the ATPmolecules in (ATP)2–FME complex and, thus, a decreaseof the local electric field exerted on the FME chromophore.

Application: Detection of endogenous formation of ATP in

rat liver mitochondria

The important fluorescence response (in terms of bothfluorescence quantum yield and bathochromic shift of theexcitation spectrum) of FME in the presence of ATP inaqueous buffered solutions prompted us to use this probefor measuring the ATP concentration changes in the organ-elles of live cells. A preliminary trial with mitochondriafrom rat liver cells, where ATP production was triggeredthrough the Krebs cycle by the succinate anion, did notallow us to observe the production of endogenous ATP[39]. Only the addition of a large amount of exogenousATP could be evidenced in these conditions. The inabilityto monitor endogenous ATP was likely related to the highconcentration of probe that did not allow the formation ofsignificant amounts of the highly fluorescent (ATP)2–FMEcomplex. In the current work, the concentration of FMEprobe was decreased 50 times and the sequence of additionof reactants was modified. We added mitochondria dis-persed in 250 mM sucrose and 10 mM Hepes (pH 7.4) tothe same incubation medium containing 0.1 lM FMEprobe. In these conditions, FME displays an excitationspectrum with its maximum at 400 nm and a small shoul-der around 470 nm (Fig. 5A). The blue shift of this excita-tion spectrum, with respect to that in aqueous sucrosesolution, suggests that part of the FME molecules may par-tition in the mitochondrial membrane [15–17,35]. The addi-tion of 6 mM succinate anion provokes a sharp andimmediate increase of the fluorescence intensity (Fig. 5B).Moreover, a new maximum in the fluorescence excitationspectrum appears at 470 nm (Fig. 5A) in line with theformation of the FME anionic form resulting from the

Page 6: Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

Fig. 5. Fluorescence excitation (A) and emission (B) spectra of FMEprobe in the presence of mitochondria before (—) and after ( ) theaddition of sodium succinate. (C) Kinetics of FME fluorescence intensityin aqueous sucrose solution. The arrows indicate the sequential addition ofmitochondria (1) and sodium succinate (2). Excitation and emissionwavelengths are 475 and 545 nm, respectively. Incubation mediumcontained 10 mM Hepes (pH 7.4), 250 mM sucrose, and 0.1 lM FME.The concentration of mitochondria was 0.15 mg/ml, and that of succinatewas 6 mM. arb.u., arbitrary units.

Sensing of ATP anion / D.A. Yushchenko et al. / Anal. Biochem. 369 (2007) 218–225 223

interaction with ATP. As shown previously [39], such aspecific effect is caused by the ATP anion only withoutany contribution from the succinate anion. Thus, theincrease of the fluorescence intensity, together with themodification of the fluorescence excitation spectra, clearlyappears to be the consequence of the formation of the(ATP)2–FME complex and can be coupled with the endog-enous production of ATP in mitochondria.

On a kinetic point of view, the addition of mitochondriato FME provokes an immediate (within 1 s) 20-foldincrease of fluorescence intensity (Fig. 5C, point 1), demon-strating a rapid penetration of the FME probe into both

the membrane and the inner space of mitochondria. Fur-ther addition of succinate anion to mitochondria provokesa slower (within 20 s) additional 5-fold increase of fluores-cence intensity (Fig. 5C, point 2) that we ascribe to theendogenous production of ATP in mitochondria throughthe enzymatic cycle induced by succinate. Thus, FMEappears to be a potential promising fluorophore for thedesign of an ATP sensor for cellular applications.

Conclusion

In this work, we have demonstrated the formation ofcomplexes between the tetraanion ATP and the flavonederivative FME. Two kinds of complexes are evidenced.The higher affinity 1:1 complex corresponds to a stackedconfiguration between the aromatic moieties of the twomolecules and leads to a strong hypochromicity of theabsorption spectrum of the dye. The lower affinity 2:1 com-plex results in a strong increase of the fluorescence intensity(�20-fold), due mainly to the appearance of the anionicform of FME, as shown by the important red shift(60 nm) of both excitation and emission spectra. Molecularmodeling indicates that this anionic form results from thedeprotonation induced by the phosphate group of the sec-ond ATP molecule. In the presence of 250 mM sucrose, theinteraction with the second ATP molecule appears to beweakened but is still appreciable. This strong fluorescenceenhancement due to the formation of the 2:1 complexshould enable the quantitative evaluation of the ATP con-centration in the physiological range, and a first set ofexperiments shows the proof of principle for the use ofFME to monitor the production of endogenous ATP inmitochondria. In this respect, we believe that FME is aninteresting basis for the design of efficient ATP sensors incells. We are currently developing new hydroxyflavonederivatives for this approach.

Acknowledgments

This work was supported by CNRS, Universite LouisPasteur, and the DNIPRO Program from the Ministeredes Affaires Etrangeres between France and Ukraine.D.A.Y. is a member of the European Doctoral Collegeand was supported by an Eiffel fellowship.

References

[1] R.P. Haugland, The Handbook: A Guide to Fluorescent Probes andLabelling Technologies, 11th ed., Invitrogen, Carlsbad, CA, 2006.

[2] G. Grynkiewicz, M. Poenie, R. Tsien, A new generation of fluores-cence Ca2+ indicators with greatly improved fluorescence properties,J. Biol. Chem. 260 (1985) 3440–3450.

[3] M.W. Hosseini, Multiple molecular recognition and catalysis: Amultifunctional anion receptor bearing an anion binding site, anintercalator group, and a catalytic site for nucleotide binding andanalysis, J. Am. Chem. Soc. 112 (1990) 3896–3904.

[4] M.E. Huston, E.U. Akkaya, A.W. Czarnik, Chelation enhancedfluorescence detection of non-metal ions, J. Am. Chem. Soc. 111(1989) 8735–8737.

Page 7: Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

224 Sensing of ATP anion / D.A. Yushchenko et al. / Anal. Biochem. 369 (2007) 218–225

[5] M.T. Albelda, M.A. Bernardo, E. Garcia-Espana, M.L. Godino-Salido, S.V. Luis, M.J. Melo, F. Pina, C. Soriano, Thermodynamicsand fluorescence emission studies on potential molecular chemosen-sors for ATP recognition in aqueous solution, J. Chem. Soc. PerkinTrans. 11 (1999) 2545–2549.

[6] J.Y. Kwon, N.J. Singh, H.N. Kim, S.K. Kim, K.S. Kim, J. Yoon,Fluorescent GTP-sensing in aqueous solution of physiological pH, J.Am. Chem. Soc. 126 (2004) 8892–8893.

[7] S.K. Kim, B-S. Moon, J.H. Park, Y.I. Seo, H.S. Koh, Y.J. Yoon,K.D. Lee, Y. Yoon, A fluorescent cavitand for the recognition ofGTP, Tetrahedron Lett. 46 (2005) 6617–6620.

[8] P.P. Neelakandan, M. Hariharan, D. Ramaiah, A supramolecularON–OFF–ON fluorescence assay for selective recognition of GTP, J.Am. Chem. Soc. 128 (2006) 11334–11335.

[9] T.D. White, Release of ATP from a synaptosomal preparation byelevated extracellular K+ and by veratridine, J. Neurochem. 30 (1978)329–336.

[10] F.R. Leach, ATP determination with firefly luciferase, J. Appl.Biochem. 3 (1981) 473–481.

[11] R.D. Beigi, G.R. Dubyak, Endotoxin activation of macrophages doesnot induce ATP release and autocrine stimulation of P2 nucleotidereceptors, J. Immunol. 165 (2000) 7189–7198.

[12] A.S. Klymchenko, T. Ozturk, V.G. Pivovarenko, A.P. Demchenko, A3-hydroxychromone with dramatically improved fluorescence prop-erties, Tetrahedron Lett. 42 (2001) 7967–7970.

[13] A.P. Demchenko, A.S. Klymchenko, V.G. Pivovarenko, S. Ercelen,Ratiometric probes: Design and applications, in: R. Kraayenhof,A.J.W.G. Visser, H.C. Gerritsen (Eds.), Fluorescence Spectroscopy,Imaging, and Probes: New Tools in Chemical, Physical, and Lifeciences, vol. 2, Springer-Verlag, Heidelberg, Germany, 2002, pp.101–110.

[14] S. Ercelen, A.D. Roshal, A.P. Demchenko, A.S. Klymchenko,Excited-state proton transfer reaction in a new benzofuryl 3-hydrox-ychromone derivative: The influence of low-polar solvents, Polish J.Chem. 76 (2002) 1287–1299.

[15] S. Ercelen, A.S. Klymchenko, A.P. Demchenko, An ultrasensitivefluorescent probe for hydrophobic range of solvent polarities, Anal.Chim. Acta 464 (2002) 273–287.

[16] A.S. Klymchenko, V.G. Pivovarenko, T. Ozturk, A.P. Demchenko,Modulation of the solvent-dependent dual emission in 3-hydroxychr-omones by substituents, New J. Chem. 27 (2003) 1336–1343.

[17] A.S. Klymchenko, V.G. Pivovarenko, A.P. Demchenko, Eliminationof hydrogen bonding effect on the solvatochromism of 3-hydroxyf-lavones, J. Phys. Chem. A 107 (2003) 4211–4216.

[18] A.S. Klymchenko, A.P. Demchenko, Multiparametric probing ofintermolecular interactions with fluorescent dye exhibiting excitedstate intramolecular proton transfer, Phys. Chem. Chem. Phys. 5(2003) 461–468.

[19] J. Guharay, R. Chaudhuri, A. Chakrabarti, P.K. Sengupta, Excitedstate proton transfer fluorescence of 3-hydroxyflavone in modelmembranes, Spectrochim. Acta A 53 (1997) 457–462.

[20] O.P. Bondar, V.G. Pivovarenko, E.S. Rowe, Flavonols: Newfluorescent membrane probes for studying the interdigitation of lipidbilayers, Biochim. Biophys. Acta 1369 (1998) 119–130.

[21] M. Sarkar, P. Sengupta, Influence of different micellar environmentson the excited-state proton transfer luminescence of 3-hydroxyflav-one, Chem. Phys. Lett. 179 (1991) 68–72.

[22] V.G. Pivovarenko, A.V. Tuganova, A.S. Klymchenko, A.P.Demchenko, Flavonols as models for fluorescent membrane probes:I. The response to the charge of micelles, Cell. Mol. Biol. Lett. 2(1997) 355–364.

[23] S.M. Dennison, J. Guharay, P.K. Sengupta, Intramolecular excited-state proton transfer and charge transfer fluorescence of a 3-hydroxyflavone derivative in micellar media, Spectrochim. Acta A55 (1999) 903–909.

[24] A.S. Klymchenko, A.P. Demchenko, Probing AOT reverse micelleswith two-color fluorescence dyes based on 3-hydroxychromone,Langmuir 18 (2002) 5637–5639.

[25] A.S. Klymchenko, A.P. Demchenko, Electrochromic modulation ofexcited-state intramolecular proton transfer: The new principle indesign of fluorescence sensors, J. Am. Chem. Soc. 124 (2002) 12372–12379.

[26] A.D. Roshal, A.V. Grigorovich, A.O. Doroshenko, V.G. Piv-ovarenko, A.P. Demchenko, Flavonols and crown-flavonols as metalcation chelators: The different nature of Ba2+ and Mg2+ complexes, J.Phys. Chem. A 102 (1998) 5907–5914.

[27] A.D. Roshal, A.V. Grigorovich, A.O. Doroshenko, V.G. Piv-ovarenko, A.P. Demchenko, Flavonols as metal-ion chelators:Complex formation with Mg2+ and Ba2+ cations in the excited state,J. Photochem. Photobiol. A 127 (1999) 89–100.

[28] X. Poteau, G. Saroja, C. Spies, R.G. Brown, The photophysics ofsome 3-hydroxyflavone derivatives in the presence of protons, alkalimetal, and alkaline earth cations, J. Photochem. Photobiol. A 162(2004) 431–439.

[29] G. Duportail, A. Klymchenko, Y. Mely, A. Demchenko, Neutralfluorescence probe with strong ratiometric response to surfacecharge of phospholipid membranes, FEBS Lett. 508 (2001) 196–200.

[30] G. Duportail, A. Klymchenko, Y. Mely, A.P. Demchenko, On thecoupling between surface charge and hydration in biomembranes:Experiments with 3-hydroxyflavone probes, J. Fluoresc. 12 (2002)181–185.

[31] A.S. Klymchenko, G. Duportail, T. Ozturk, V.G. Pivovarenko, Y.Mely, A.P. Demchenko, Novel two-band ratiometric fluorescenceprobes with different location and orientation in phospholipidmembranes, Chem. Biol. 9 (2002) 1199–1208.

[32] A.S. Klymchenko, G. Duportail, T. Ozturk, Y. Mely, A.P. Dem-chenko, Ultrasensitive two-color fluorescence probes for dipolepotential in phospholipid membranes, Proc. Natl. Acad. Sci. USA100 (2003) 11219–11224.

[33] A.S. Klymchenko, H. Stoeckel, K. Takeda, Y. Mely, Fluorescentprobe based on intramolecular proton transfer for fast ratiometricmeasurement of cellular transmembrane potential, J. Phys. Chem. B110 (2006) 13624–13632.

[34] N.A. Nemkovich, J.V. Kruchenok, A.N. Rubinov, V.G. Piv-ovarenko, W. Baumann, Site selectivity in excited-state intramolec-ular proton transfer in flavonols, J. Photochem. Photobiol. A 139(2001) 53–62.

[35] N.A. Nemkovich, W. Baumann, V.G. Pivovarenko, Dipole momentsof 4’-aminoflavonols determined using electro-optical absorptionmeasurements or molecular Stark-effect spectroscopy, J. Photochem.Photobiol. A 140 (2002) 19–24.

[36] N.A. Nemkovich, W. Baumann, V.G. Pivovarenko, A.N. Rubinov,Determination of the dipole moments of the molecules of 4’-substituted 3-hydroxyflavones using the electrooptic absorptionmethod, J. Appl. Spectr. 70 (2003) 230–237.

[37] L.V. Premvardhan, L.A. Peteanu, Dipolar properties of andtemperature effects on the electronic states of 3-hydroxyflavone(3HF) determined using Stark-effect spectroscopy and comparedto electronic structure calculation, J. Phys. Chem. A 103 (1999)7506–7514.

[38] A.S. Klymchenko, V.G. Pivovarenko, A.P. Demchenko, Perturbationof planarity as the possible mechanism of solvent-dependent varia-tions of fluorescence quantum yield in 2-aryl-3-hydroxychromones,Spectrochim. Acta A 59 (2003) 787–792.

[39] V.G. Pivovarenko, O.B. Vadzyuk, S.O. Kosterin, Fluorometricdetection of adenosine triphosphate with 3-hydroxy-4 0-(dimeth-ylamino)flavone in aqueous solutions, J. Fluoresc. 16 (2006)9–15.

[40] S.M. Ormson, R.G. Brown, F. Vollmer, W. Rettig, Switchingbetween charge- and proton-transfer emission in the excited state ofa substituted 3-hydroxyflavone, J. Photochem. Photobiol. A 81 (1994)65–72.

[41] M.J.S. Dewar, E.J. Zoebisch, E.F. Healy, J.J.P. Stewart, AM1: A newgeneral purpose quantum mechanical molecular model, J. Am. Chem.Soc. 107 (1985) 3902–3908.

Page 8: Sensing of adenosine-5′-triphosphate anion in aqueous solutions and mitochondria by a fluorescent 3-hydroxyflavone dye

Sensing of ATP anion / D.A. Yushchenko et al. / Anal. Biochem. 369 (2007) 218–225 225

[42] A.O. Doroshenko, Spectral Data Lab [computer software], Kharkiv,Ukraine, 1999.

[43] D.M. Himmelblau, Applied Nonlinear Programming, McGraw–Hill,New York, 1972.

[44] C.R. Cantor, P.R. Schimmel, Biophysical Chemistry, in: W.H.Freeman (Ed.), San Francisco, 1980.

[45] V.V. Shynkar, A.S. Klymchenko, Y. Mely, G. Duportail, V.G.Pivovarenko, Anion formation of 4(-(dimethylamino)-3-hydroxyflav-one in phosphatidylglycerol vesicles induced by Hepes buffer: A

steady-state and time-resolved fluorescence investigation, J. Phys.Chem. B 108 (2004) 18750–18755.

[46] D. Yuan, R.G. Brown, Enhanced nonradiative decay inaqueous solutions of aminonaphthalimide derivatives viawater-cluster formation, J. Phys. Chem. A 101 (1997)3461–3466.

[47] S. Tobita, K. Ida, S. Shiobara, Water-induced fluorescence quenchingof aniline and its derivatives in aqueous solution, Res. Chem.Intermed. 27 (2004) 205–218.