A study of the intermolecular interactions of tolmetin/N-acetyl-l-tyrosine ethyl ester complex

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Spectrochimica Acta Part A 72 (2009) 1000–1006

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

study of the intermolecular interactions of tolmetin/N-acetyl-l-tyrosinethyl ester complex

ntonio Titoa,∗, Claudia Jimenez-Lopeza, Agnieszka Kowalskab, Stanisław Wysockib

Natural Science Department, University of Houston-Downtown, One Main Street, Houston, TX 77002, USADepartment of Biotechnology and Food Science, Lodz University of Technology, ul. Stefanowskiego 4/10, PL 90-924, Lodz, Poland

r t i c l e i n f o

rticle history:eceived 19 August 2008

a b s t r a c t

The formation of tolmetin/N-acetyl-l-tyrosine ethyl ester (ATEE) complex has been reported by means of

ccepted 18 December 2008

eywords:ime-resolved fluorescence spectroscopyolmetinuantum-mechanical calculations

both theoretical and experimental studies, including quantum mechanical calculations as well as UV–visabsorption, fluorescence and time-resolved spectroscopy measurements. It has been found that the flu-orescence of ATEE is quenched due to the formation of a non-fluorescent complex between ATEE andtolmetin in the ground state. The geometrical parameters of ATEE/tolmetin complex have been deter-mined with the use of the DFT method applying the B3LYP correlation-exchange functional and 6-31G(d)basis set. The results of experiments indicated the static ATEE quenching by tolmetin. Additionally, the

tically

experimental and theore

. Introduction

Most of the drugs used for the treatment of inflammationre carboxylic acids in which the carboxylate group is avail-ble for metal–ligand interactions. Some of these drugs belongo the group of anti-inflammatory drugs known as non-steroidnti-inflammatory drugs (NSAIDs) [1]. Tolmetin [1-methyl-5-(4-ethylbenzoyl)-1H-pyrrole-2-acetic acid] is a NSAIDs of the family

f the arylalkanoic acids [2].Tolmetin exhibits anti-inflammatory, analgesic and antipyretic

roperties. In addition, this drug is used for the treatment ofnflammation and pain that results from muscle skeletal and boneelated diseases such as rheumatoid arthritis, juvenile arthritis, andsteoarthritis [3].

The role of tolmetin, as many others NSAIDs, is to inhibityclooxygenases (COX) enzymes which have an active role in theroduction of prostaglandins. There are two isoforms of cyclooxy-enase: COX 1 and COX 2. COX 1 catalyzes the normal productionf prostaglandins in the body while COX 2 is involved in the pro-uction of prostaglandins in inflammatory cells [4]. COX 1 containswo active sites, including, a cyclooxygenase and a peroxidase.OX1 catalyzes the production of prostaglandins by removing

hydrogen atom from arachidonic acid and transferring it to

yr385 in the active site of COX1. A hydrogen bond betweenyr348–Tyr385 is essential for this activity, thus, any disruptionould alter prostaglandins synthesis. Moreover, it is believed that

∗ Corresponding author. Tel.: +1 281 920 1717; fax: +1 281 920 1717.E-mail address: [email protected] (A. Tito).

386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2008.12.034

predicted Gibbs free energy of complexation has been calculated.© 2008 Elsevier B.V. All rights reserved.

in order to inhibit prostaglandins production, tolmetin has to inter-act with tyrosine in the active site of COX1 to block its catalyticactivity [5].

Due to the role that tyrosine residues play in the biological activ-ity of COX1, tyrosine has also become the main object of this study.

Tyrosine is one of the 20 basic biogenic amino acids and it isextremely reactive due to its hydrophilic side chain. Tyrosine can beregarded as a derivative of phenylethylamine because its precursoris known as phenylalanine with a hydroxyl group in “-para” posi-tion in the aromatic ring. In addition, tyrosine is an essential aminoacid because it is needed for intracellular transport, synthesis ofhormones and biologically active substances such as adrenaline,noradrenalin, and dopamine [6]. Tyrosine is the a large constituentof all residues at the active site of cyclooxygenase as well as enzymessuch as topoisomerases. Thus, tyrosine will most likely interact withany drug that targets the enzyme’s active site. In fact, tyrosine inter-acts with the phosphoryl group of DNA during the replication phaseof cell division, which is exacerbated during cancer [7].

In this project, N-acetyl-LTyrosine ethyl ester has been used asa derivatized analog of amino acid tyrosine. Tyrosine was esterifiedto ATEE because in peptides only the phenol group is available forinteractions.

The goal of this investigation is to quantitatively describe theinteractions between tolmetin and ATEE in ground and excitedstates. In order to carry out this study, both experimental and com-

putational methods have been applied. Experimental techniquesof UV absorption, fluorescence and time-resolved fluorescencespectroscopies have been used to determine the nature of the inter-actions between tolmetin and ATEE. The geometry optimizationand frequencies calculations for tolmetin, ATEE and ATEE/tolmetin

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A. Tito et al. / Spectrochimica

omplex have been performed applying the DFT(B3LYP)/6-31G(d)ethod. The geometrical parameters of ATEE/tolmetin complex in

he ground state have been determined. The calculated spectro-copic parameters of OH and CH stretching vibrations in isolatedTEE molecule have been compared with the corresponding val-es in the ATEE/tolmetin complex to analyze their shifts due to theomplex formation. Finally, the theoretically predicted Gibbs freenergy of association has been compared with the experimentallybtained value.

. Experimental and computational methods

.1. Materials

Tolmetin and N-acetyl-l-tyrosine ethyl ester (ATEE) producedy Sigma were dissolved in 0.1 M phosphate buffer (pH 7) and inater, respectively.

All reagents were at least analytical grade and were used withouturther purification.

.2. Experimental methods

The absorption spectra were record using A Nicolet Evolution00 UV-Vis spectrophotometer instrument from Thermo Electronorporation.

The steady-state fluorescence measurements were performedsing a Fluoromax-2 from Jobin Yvon-Spex. The solutions for flu-rescence and absorption titration experiments were prepared byarying the concentration of tolmetin (0–9.38 × 10−6) and keepinghe concentration of ATEE constant (6 × 10−6 M). A concentrationf ∼10−6 M for ATEE and tolmetin solutions was applied to avoidhe inner-filter effect in the fluorescence spectra.

The concentration range of phosphate buffer in solutions usedor absorption and fluorescence titration experiments was from 0

o 9.38 × 10−4 M.

Fluorescence decays were obtained using the time-correlatedingle-photon-counting method (Edinburgh Instruments OB-920).he excitation source was a hydrogen nanosecond flash lamp with aepetition rate of 40 kHz. Fluorescence decays were analyzed using

Fig. 1. Absorption spectr

art A 72 (2009) 1000–1006 1001

F-900 Spectrometer Software. All experiments were performed at25 ◦C.

2.3. Computational methods

All calculations were carried out using HyperChem 7.0 andGaussian 03 programms. The geometries of tolmetin, ATEE aswell as the tolmetin/ATEE complex were optimized in the groundstate using molecular mechanics (UFF), semi-empirical (AM1), andthe DFT method with the B3LYP correlation-exchange functionwith 6-31G(d) basis set in vacuum. In order to make sure thatthe DFT(B3LYP)/6-31G(d) optimized structures correspond to theenergy minimum, frequencies calculations were also performed atthe same theoretical level. No negative frequency was obtainedwhich indicated that the optimized structures represented localminima on the DFT(B3LYP)/6-31G(d) electronic energy surface.The interaction energy between tolmetin and ATEE at the ener-getic minimum was calculated using the super-molecule approach,which defines it as the difference between the electronic energyof the complex and the sum of electronic energy of the isolatedmonomers. The Gibbs free energy was calculated from the thermo-dynamic data listed in the output of Gaussian calculations withoutscaling in vibrational frequencies. In addition, the harmonic approx-imation was applied in evaluating the vibrational contributions.

3. Results and discussion

3.1. Absorption spectra

The UV absorption spectra of ATEE dissolved in water are shownin Fig. 1. The spectra consist of two peaks situated at about 229 and275 nm. The UV absorption spectra of tolmetin (shown in Fig. 2)dissolved in phosphate buffer (pH 7) consist of three peaks situatedat about 201, 260 and 325 nm.

The obtained absorption spectra of tolmetin and ATEE are con-sistent with previously reported data [8,9]. The linearity of theabsorbance upon increasing concentration of ATEE and tolmetinsuggests no dimerisation reactions occur between ATEE and tol-metin molecules in the ground state. This applies for the studied

a of ATEE in water.

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olmet

cm

iacatt

ma

3

rfl

Fig. 2. Absorption spectra of t

oncentration range of ATEE (3 × 10−5 M to 1 × 10−3 M) and tol-etin (5 × 10−6 M to 1.1 × 10−4 M).The variations in the absorption spectra of ATEE upon increas-

ng concentration of tolmetin solution are shown in Fig. 3. Nodditional band associated with the formation of ATEE/tolmetinomplex is observed upon increasing concentration of tolmetin. Thebsorption spectra for ATEE, tolmetin and the mixture of ATEE andolmetin are plotted in Fig. 4 together with the calculated sum ofhe absorbance of ATEE and the absorbance of tolmetin.

It can be seen that the absorption spectra of the ATEE and tol-etin mixture show a maxima situated at the same wavelengths

s for the calculated sum of ATEE and tolmetin absorbances.

.2. Fluorescence spectra

It is known that phosphate and other ions quench tyrosine fluo-escence [10–12]. To examine the influence of phosphate ions on theuorescence spectra of ATEE, we have recorded emission spectra of

Fig. 3. Absorption spectra of ATEE (6 × 10−6 M) upon incre

in in phosphate buffer (pH 7).

ATEE (6 × 10−5 M) in water upon adding increasing concentrationof phosphate buffer (the concentration range of phosphate bufferused is from 0 to 2.06 × 10−3 M). The fluorescence spectra of ATEEupon adding phosphate buffer (0–2.06 × 10−3 M) are presented inFig. 5.

The results suggest that the concentration range of phosphatebuffer used in this study has no or little effect on the fluorescenceintensity of ATEE (Fig. 5).

The emission spectra of ATEE (6 × 10−6 M) upon increasing con-centration of tolmetin excited at 278 nm are shown in Fig. 6. Themaximum concentration of phosphate ions in solutions used forthe fluorescence titration experiments was up to 9.38 × 10−4 M.It can be seen that only one type of emission with a maximum

at 306 nm for ATEE occurs which is consistent with the previ-ously reported data [13]. Moreover, the fluorescence of ATEE isquenched upon increasing concentration of tolmetin. The spectrado not show a new peak which suggests that ATEE/tolmetin com-plex is non-fluorescent. Additionally, the fluorescence excitation

asing concentration of tolmetin ((0–9.3)8 × 10−6 M).

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Fig. 4. Absorption spectra of ATEE (6 × 10−5 M), tolmetin (5 × 10−6 M) and the mixture of ATEE and tolmetin.

Fig. 5. Fluorescence spectra of ATEE (6 × 10−5 M) excited at 278 nm upon increasing concentration of phosphate buffer (0–2.06 × 10−3 M).

Fig. 6. Fluorescence spectra of ATEE (6 × 10−6 M) excited at 278 nm upon increasing concentration of tolmetin ((0–9.38) × 10−6 M).

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1004 A. Tito et al. / Spectrochimica Acta Part A 72 (2009) 1000–1006

04 nm

sassipn

re

wflkooet

qAetwo

3

tflatt(cg

aA

Fi = ˛˙i˚iεiCi (4)

where ˛, apparatus factor; ˚i, fluorescence quantum yield of i; εi,molar extinction coefficient of i; Ci, concentration of i; Eq. (4) may

Table 1Fluorescence decay times (�1 and �2) and pre-exponential factors (B1 and B2) ofATEE upon increasing concentration of tolmetin. Excitation at 278 nm, emission at304 nm. The average fluorescence decay time (�) was calculated from the formula:� = ((�2

1 B1) + (�22 B2))/((�1B1 + �2B2)).

ATEE Tolmetin �1 �2 � [ns]

[ns] B1 [ns] B2

6 × 10−5 0 1.0187 0.074 3.2413 0.001 1.116 × 10−5 5 × 10−6 1.0501 0.073 3.8915 0.001 1.19

Fig. 7. Fluorescence excitation spectra of ATEE (6 × 10−6 M) at 3

pectra graph of ATEE/tolmetin solutions shows the same shapes for ATEE solution (Fig. 7). The shape of fluorescence excitationpectra for ATEE–tolmetin solutions does not depend on the emis-ion wavelength which suggests that ATEE is the only fluorophoren the ATEE/tolmetin systems. The latter confirms that the com-lex formed between ATEE and tolmetin in the ground state ison-fluorescent.

It is known that the quenching mechanism of fluorophore fluo-escence by a quencher is described by the following Stern–Volmerquation:

F0

Fi= (1 + kq�0[Qi])(1 + K[Q0]) (1)

here F0, fluorescence intensity in the absence of a quencher; Fi,uorescence intensity for changing concentration of a quencher;q, rate constant for quenching; �0, lifetime of fluorescence decayf chromophore in the absence of a quencher; [Qi], concentrationf a free quencher; [Q0], analytical concentration of a quencher; K,quilibrium constant for complex formation in the ground state, inhe presence of a quencher.

In this equation, the first set of parentheses describes a dynamicuenching while the second set describes a static quenching.ccording to Nevin et al. [13], the fluorescence quenching is due toither a non-radiative deactivation on the excited state or an inhibi-ion of the excited state formation. These two scenarios determinehether the static, dynamic or combined mechanism of quenching

ccurs [14,15].

.3. Time-resolved fluorescence

In order to get insight into the quenching mechanism of ATEE byolmetin, time resolved fluorescence spectroscopy was used. Theuorescence decay of ATEE in water (excitation at 278 nm, emissiont 304 nm) is bi-exponential with the average fluorescence life-ime of 1.11 ns (Table 1). Upon increasing concentration of tolmetinhe fluorescence decay time of ATEE does not change significantlyFig. 8). This suggests a static quenching of ATEE by tolmetin, indi-

ating that the complex between ATEE and tolmetin forms in theround state.

In our experiment the concentrations of the fluorophorend the quencher are comparable ([Q0]≈[A0]). The formation ofTEE/tolmetin complex in the ground state may be described by

upon increasing concentration of tolmetin ((0–9.38) × 10−6 M).

the reaction:

[A] + [Q] ↔ [AQ]

where A denotes ATEE, Q denotes the quencher (tolmetin), and AQ-complex between ATEE and tolmetin. From the definition of theequilibrium constant for the above reaction and the mass law, thefollowing equation is obtained:

K = CAQ

CACQ= CAQ

(CoA − CAQ )(Co

Q − CAQ )(2)

where CA, CQ, and CAQ are the equilibrium concentrations of ATEE(A), tolmetin (Q) and ATEE/tolmetin (AQ) complex, respectively;C0

A , and C0Q are the initial (analytical) concentrations of ATEE and

tolmetin, respectively.The acceptable physical root of this equation gives CAQ:

CAQ = 12

{(CO

Q + COA + 1

K

)−

√(CO

Q + COA + 1

K

)2− 4CO

Q COA

}(3)

CAQ from Eq. (3) can be substituted into Eq. (2) in order to calculateK.

Because:

6 × 10−5 1 × 10−5 1.0570 0.072 3.6467 0.001 1.186 × 10−5 2 × 10−5 1.0077 0.072 3.0558 0.002 1.176 × 10−5 3 × 10−5 1.0422 0.071 3.3488 0.002 1.236 × 10−5 5 × 10−5 1.0280 0.072 3.4493 0.002 1.236 × 10−5 7 × 10−5 0.9783 0.069 3.0306 0.003 1.22

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F ay tim� tolme

b

F

�c

F

F

t

F

scb

Itoi

Ff

K

wtK(

3

cb

quencies may provide very useful information about the strength ofthe interactions involved in ATEE/tolmetin hydrogen-bonded com-plex. Upon the formation of a hydrogen bond it is expected thatstretching vibration involving the proton-donating group should

ig. 8. The influence of increasing concentration of tolmetin on the fluorescence decis the lifetime of ATEE fluorescence in the presence of increasing concentration of

e written as follows:

i = ˛(˚AεACA + ˚Q εQ CQ + ˚AQ εAQ CAQ ) (5)

From the experimental conditions (�exc = 278 nm,em = 304 nm), it can be seen that ˚Q = 0 and ˚AQ = 0. In thisase Eq. (5) has the form:

i = ˛˚AεACA (6)

Considering CA = C0A − CAQ Eq. (6) has the following form:

i = ˛˚AεA(C0A − CAQ ) (7)

The intensity of fluorescence emission without tolmetin is equalo

0 = ˛˚0AεAC0

A (8)

Because the fluorescence decay time of ATEE does not changeignificantly upon increasing concentration of tolmetin it can beoncluded that ˚0

A = ˚A. Therefore the ratio F0/Fi can be describedy the formula:

F0

Fi= ˛˚AεAC0

A

˛˚AεA(C0A − CAQ )

= C0A

C0A − CAQ

(9)

t is possible to determine K when CAQ calculated from Eq. (3) andhe experimental data (F0/Fi) are considered into Eq. (9). The graphbtained by fitting the experimental data to Eq. (9) using the Orig-nPro7.5 program is shown in Fig. 9.

In this way obtained K value is equal to 3.33 × 104 M−1 (Fig. 9).rom this K value the Gibbs free energy may be calculated from theollowing formula:

= exp(

−�G

RT

)(10)

here R is the universal gas constant (8314 J/(mol K)) and T ishe absolute temperature (298.15 K). The calculation of �G withusing a rearrangement of Eq. (10), gives a value of −25,815 kJ/mol

−6170 kcal/mol).

.4. Theoretical calculations

The DFT(B3LYP)/6-31G(d) optimized structure of ATEE/tolmetinomplex shown in Fig. 10 reveals the presence of a strong hydrogen-onded interaction between the ATEE phenol –OH group and the

e of ATEE. �0 is the lifetime of ATEE fluorescence in the absence of tolmetin, whereastin.

carboxylic oxygen of tolmetin. The results of calculations indi-cate that this hydrogen bond is nearly linear (<OH· · ·O = C is 167◦)and the distance between proton donor (ATEE oxygen atom of OHgroup) and proton acceptor (carboxylic oxygen of tolmetin) is equalto 2.62 Å. The complex is stabilized by the presence of anotherhydrogen bond formed between tolmetin carboxylic group and thehydrogen atom which belongs to the phenol ring of ATEE. The cal-culated distance between atoms involved in the formation of theabove mentioned hydrogen bond is 3.28 Å, whereas the < CH· · ·Oangle is calculated to be 167◦. Both distances and angles indi-cate that the geometrical criteria for hydrogen bonds are fulfilled.The longer distance between proton donor and proton acceptoratoms for the second hydrogen bond indicates that the strengthof this interaction is weaker in comparison with the strength of theinteraction between carboxylic oxygen atom of tolmetin and ATEEphenol OH group.

The DFT(B3LYP)/6-31G(d) calculated harmonic vibrational fre-

Fig. 9. Fitting the experimental data (F0/Fi) to Eq. (9).

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1006 A. Tito et al. / Spectrochimica Acta Part A 72 (2009) 1000–1006

Fig. 10. The DFT(B3LYP)/6-31G(d) optimized structure of ATEE/tolmetin complex.

Table 2The DFT(B3LYP)/6-31G(d) calculated spectroscopic parameters of OH and CH stretching vibrations and its changes due to the complex formation.

ICOH

A –A 89

stcc

auCsuctAi

2−

4

aaaoAcDmhAons

(vfi

cpctmb

[[

[[

�OH [cm−1] IOH ��OH [cm−1]

TEE 3751.31 43.36 –TEE/tolmetin 2916.88 3886.14 −834.43

how shift in its frequency towards lower energy. Additionally,he intensity of the proton-donating group stretching vibration inomplex should be enhanced significantly in comparison with thatorresponding to the isolated molecule [16].

Table 2 collects the calculated spectroscopic parameters for OHnd CH stretching frequencies in ATEE/tolmetin complex. These val-es have been compared to those corresponding to the OH andH stretching frequencies in isolated ATEE. It can be seen that thetretching OH and CH frequencies associated with hydrogen bondsndergo red-shift to lower frequencies compared with the frequen-ies corresponding to the free monomer (ATEE). This occurs becausehe formation of the hydrogen bond weakens the OH or CH bond.dditionally, it can be seen that the stretching HO and CH intensities

nvolved in the hydrogen bonds are enhanced largely.The Gibbs free energy of association (�G) computed at

98.15 K and the pressure of 1 atm in vacuum is calculated to be49,815 kJ/mol (−11,906 kcal/mol).

. Conclusions

In this study, the intermolecular interactions between ATEEnd tolmetin were investigated using both experimental (UV–visbsorption, steady-state and time-resolved fluorescence) as wells theoretical methods (quantum mechanical calculations). Thebtained results indicated the formation of the non-fluorescentTEE/tolmetin complex in the ground state. The geometri-al parameters of the complex were determined using theFT(B3LYP)/6-31G(d) method. Inspection of the optimized geo-etrical parameters of the complex revealed the presence of two

ydrogen bonds interactions. One of them was strong and involvedTEE phenol group and tolmetin carboxylic oxygen. The presencef the second hydrogen bond between hydrogen atom of ATEE phe-ol ring and carboxylic oxygen of tolmetin resulted in additionaltabilization of the complex.

The DFT(B3LYP)/6-31G(d) calculated �G of association−11,906 kcal/mol) did not correlate well with experimentalalue (−6170 kcal/mol). The expected inconsistency might resultrom the fact that no solvent effects were taken into considerationn the applied theoretical model.

Additionally, the theoretically obtained vibrational frequen-ies and intensities of the stretching vibrations involving

roton-donating groups in the complex were compared to theorresponding values in monomers to evaluate the shifts due tohe hydrogen bond formation. It was found that the stretching

odes of OH and CH of ATEE phenol ring involved in the hydrogenonds undergo shifts toward lower frequency upon complexation.

[

[

[[

/IMOH �CH [cm−1] ICH ��CH [cm−1] IC

CH/IMCH

3186.94 17.66 – –.6 3154.66 182.36 −32.3 10.3

Moreover significant enhancement in OH and CH intensities inATEE/tolmetin complex was reported.

The results of our studies suggested the formation of the non-fluorescent ATEE/tolmetin complex in the ground state whichconfirmed previously reported data about the presence of inter-molecular interactions between ATEE and tolmetin [17]. Thepresence of intermolecular interactions between tyrosine and tol-metin may enable possible improvements of tolmetin drug to makeit more efficient in targeting tyrosine at a molecular level. Con-sequently, such drug could serve as an inhibitor of the activity ofuncontrolled tyrosine kinases receptors that lead to diseases suchas cancer.

Acknowledgements

We would like to thank the Brown Foundation Inc. for fund-ing the Research and Study Abroad program set up by the UHDNatural Science Department and TUL Department of Biotechnol-ogy and Food Sciences (PI: Dr. Uzman, Akif). We would also like tothank Dr. Larry Spears, Dr. Akif Uzman, Dr. Grebowicz, UHD NS staff,Wysocki lab members, and our families for their invaluable supportthroughout this time.

References

[1] A. Jubert, M. Leticia-Legarto, N.E. Massa, L.L. Tevez, N.B. Okulik, J. Mol. Struct.783 (2005) 34–51.

[2] http://redpoll.pharmacy.ualberta.ca/drugbank/ DrugBank Tolmetin(APRD01268) University of Alberta, Canada, 2007.

[3] N.B. Okulik, A.H. Jubert, J. Mol. Struct. 769 (2005) 135–141.[4] D.S. Goodsell, Stem Cells 18 (2000) 227–229.[5] C.E. Rogge, B. Ho, W. Liu, R.J. Kulmacz, A. Tsai, Biochemistry 45 (2006)

523–532.[6] J. Bullock, M. Wang, J. Boyle, Physiology, fourth ed., Wolters Kluwer Health, P.A.,

Philadelphia, 2001.[7] Y. Pommier, P. Pourquier, Y. Fan, D. Strumberg, Biochim. Biophys. Acta 1400

(1998) 83–106.[8] S. Chang, C.S. Chien, H. Lee, C.Y. Chen, J. Food Drug Anal. 7 (4) (1999) 259–268.[9] F.E. Ali, K.J. Barnham, C.J. Barrow, F. Sparovic, J. Inorg. Biochem. 98 (2004)

173–184.10] O. Shimizu, K. Imakubo, Photochem. Photobiol. 30 (1977) 541–543.11] T. Alev-Behmoras, J.-J. Toulme, C. Helene, Photochem. Photobiol. 30 (1979)

533–539.12] J.K. Lee, R.T. Ross, J. Phys. Chem. B 102 (1998) 4612–4618.13] A. Nevin, S. Cather, D. Anglos, C. Fotakis, Anal. Chim. Acta 573–574 (2006)

341–346.

14] A. Sharma, S.G. Schulman, Introduction to Fluorescence Spectroscopy A, Wiley-

Interscience Publication, John Wiley and Sons Inc., NY, 1999.15] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Aca-

demic, NY, 1999.16] O. Wu, H. Zhang, Z. Zhou, J. Lu, G. Zhao, J. Mol. Struct. 757 (2005) 9–18.17] A. Levitzki, A. Gazit, Science 267 (1995) 1782–1788.