Journal of Molecular Structure 927 (2009) 1–6

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Journal of Molecular Structure

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Spectrofluorimetric study of the binding of codeine to nucleic acids

Feng Wang *, Wei Huang, Liang Su, Zijia Dong, Shuai ZhangDepartment of Chemistry, Zaozhuang University, Zaozhuang 277160, PR China

a r t i c l e i n f o

Article history:Received 27 October 2008Received in revised form 2 January 2009Accepted 14 January 2009Available online 22 January 2009

Keywords:Fluorescence spectrumCodeineCalf thymus DNA (ctDNA)Yeast RNA (yRNA)

0022-2860/$ - see front matter � 2009 Published bydoi:10.1016/j.molstruc.2009.01.022

* Corresponding author. Tel.: +86 6323786735; faxE-mail addresses: wf332@uzz.edu.cn, wf3786621@

a b s t r a c t

The characteristics of the interaction between codeine (CD) and nucleic acids were studied by ultraviolet–visible spectra and fluorescent spectra. It shows that there is a powerful ability in nucleic acids to quenchthe fluorescence intensity of codeine. The fluorescence quenching data were analyzed according to Stern–Volmer equation and Förster’s nonradiative energy transfer mechanism. Thus the binding constant andthe thermodynamic parameters between codeine and nucleic acids were obtained. The results show thatcodeine interacts with nucleic acids in a mode of groove binding and –OCH3 of the codeine molecularcombines with the groove of nucleic acids through hydrogen bond or van der Waals force.

� 2009 Published by Elsevier B.V.

1. Introduction

Several small molecules have been shown to interact with DNAat the molecular level by specific binding modes. These bindingstudies have been of great interest for a long time because theirimportance in understanding the mechanism of anticancer drugaction at the molecular level and the consequent design of newefficient drugs targeted to DNA [1–5].

Generally, the noncovalent interaction of small molecules withDNA involve three binding modes: namely, intercalative bindingthat dyes intercalate into the base pairs of nucleic acids, groovebinding in which the dyes bound on nucleic acids are located inthe major or minor groove and electrostatic binding [6–7]. It hasbeen demonstrated that DNA-binding agents possess anti-tumor,anti-viral, or anti-microbial activity, and certain substances are ofpharmacological and medical importance. Except synthetic com-pounds, a number of natural products are capable of forming com-plexes with DNA. Among the biologically active molecules fromnatural sources, alkaloids occupy an important position in medici-nal chemistry due to their extensive biological activities.

Codeine (CD) is an alkaloid, an analgesic with uses similar tomorphines, usually used as the phosphate form: Codeine phos-phate (7, 8-didehydro-4, 5a-epoxy-3-methoxy-17-methylmorph-inan-6a-ol phosphate (1:1) (salt) hemihydrate) [8]. Codeine isthe most widely used, naturally occurring narcotic in medicaltreatment in the world, used in therapy for its antitussive, analge-sic and antidiarrheic effects. Compared to morphine, codeine pro-duces less analgesia, sedation, and respiratory depression.

Elsevier B.V.

: +86 6323786850.163.com (F. Wang).

The structure of codeine is as follows:

OCH3O OH

CH2

N CH3

H3PO4 H2O11

2

In this paper, the interaction between codeine and nucleic acidswere studied attempting to make clear that DNA probably is thetarget molecules of the alkaloid, and to help people further exploreits biological activity mechanisms. The investigation on the bind-ing properties of the compound with DNA was reported based onfluorescence and absorption spectra.

2. Experiment

2.1. Apparatus

Normal fluorescence measurements were recorded with a F-2500 spectrofluorimeter (Hitachi, Japan).

All pH measurements were made with a pHS-2F digital aciditymeter (Leici, Shanghai).

All absorption spectra were recorded with an UV-2401 spectro-photometer (Shimadzu, Japan).

2 F. Wang et al. / Journal of Molecular Structure 927 (2009) 1–6

2.2. Chemicals

Codeine (CD) (Chinese Pharmaceutical and Biological Test Insti-tute, Beijing): 1.0 � 10�4 mol L�1 was prepared by dissolving CD indoubly deionized water.

Stock solutions of ctDNA (500 mg L�1) and yRNA(500 mg L�1)were prepared by dissolving commercial calf thymus DNA (ctDNA)(Hua-mei Biochemical Reagent Co., China) and yeast RNA (yRNA)(Institution Biochemistry, Chinese Academy of Sciences) in water.The working solution was prepared by diluting the stock solutionto the proper concentration. The purity of DNA or RNA waschecked by measuring the ratio of the absorbance of 260 nm tothat of 280 nm.

Above solutions needed to be stored at 0–4 �C.The hexamethylene tetramine (HMTA-HCl) buffer solution

(0.10 mol L�1) was used for the pH adjustment.All the chemicals used were of analytical reagents grade and

doubly deionized water was used throughout.

2.3. Procedure

To a 10 mL test-tube, solutions were added as the following or-der: 1.00 mL HMTA-HCl (pH 7.40), 1.00 mL of CD (1.00 �10�4 mol L�1), definite standard ctDNA or yRNA. The mixture wasdiluted to 10 mL with water and allowed to stand for 20 min.The fluorescence intensity was measured in a 1 cm quartz cell atkex/kem = 280/345 nm and with slit at 10.0 nm for the excitationand emission. All the UV absorption spectra were measured by aUV-2401 spectrophotometer at the same time.

3. Result and discussion

3.1. Fluorescence quenching spectra

Codeine behaves steady state fluorescence which is obtained at345 nm when excited by the ultraviolet wavelength (k) 280 nm,while there isn’t in nucleic acids. Fluorescence spectra of codeinewere determined in the presence of increasing amount of ctDNAat room temperature, as shown in Fig. 1. It can be seen that thefluorescence of CD is quenched by ctDNA, and the little blue shiftsabout 1–2 nm of the fluorescence peak of CD is observed in thepresence of increasing ctDNA concentration, which suggestingthe chromophore is placed in a more hydrophobic environmentafter the addition of ctDNA [7]. All these indicate that ctDNA couldinteract with CD. And so does yRNA.

Fig. 1. Fluorescence quenching spectra of CD as ctDNA were added. Conditions: pH7.4; CD: 1.0 � 10�5 mol L�1; from 1 to 10: ctDNA (g L�1): 0, 0.01, 0.025, 0.04, 0.05,0.075, 0.09, 0.10, 0.125, 0.15.

3.2. Optimization of experimental conditions

3.2.1. Effect of the buffer on the fluorescence quenching extentIn the physiological acidity, pH 7.40, experiments indicate that

the different buffers have a large effect on the fluorescence inten-sity of the system. The fluorescence quenching extent for the bufferof Tris–HCl, KH2PO4–NaOH, Na2HPO4–KH2PO4, BR (Britton–Robin-son buffer) and HMTA is 96.77, 85.01, 92.89, 90.48, 87.86 and 100.The results show that HMTA is the most suitable buffer. Therefore,the 1.00 mL pH 7.40 HMTA is chosen to the further research.

3.2.2. Signal stabilityUnder the optimum condition, the effect of time on the fluores-

cence intensity was studied. The result shows that the fluorescencequenching reaches a maximum after 15 min and remains stable forover 5 h.

3.3. Fluorescence quenching measurement of DNA on the fluorescenceof CD

3.3.1. Fluorescence quenching constantFluorescence quenching refers to any process that decreases the

fluorescence intensity of a sample such as excited state reactions,energy transfers, ground-state complexes formation and collisionalprocess [9]. Collisional quenching, or dynamic quenching, resultsfrom collision between fluorophores and a quencher. Staticquenching is due to ground-state complex formation between fluo-rophores and a quencher. Collisional quenching is described byStern–Volmer equation, which gives the ratio between fluores-cence intensities in the absence or presence of a quencher as afunction of its concentratio. From this equation, the Stern–Volmeror quenching constant can be calculated, which are use to distin-guish dynamic from static quenching [10]. The quenching equationis presented by:

F0=F ¼ 1þ Ksv½Q � ð1Þ

where F and F0 are the fluorescence intensity with and withoutquencher, respectively. [Q] is the concentration of quencher and[Q] is the concentration of the ctDNA in this paper. Ksv is theStern–Volmer quenching constant and also is static quenching con-stant when it is static quenching reaction.

The quantitative analysis of the binding ctDNA-CD was carriedout using the Stern–Volmer equation at various temperatures.The plots of F0/F versus [Q] (intercept = 1, slope = Ksv) are shownin Fig. 2 and the quenching constant Ksv are listed in Table 1. Obvi-ously, the quenching constant of CD quenching procedure initiatedby ctDNA increased along with the rise of the temperature. Thismeans that the quenching is initiated by dynamic collision butnot from the formation of a complex [10].

3.3.2. Binding parametersWhen molecules binding dependently to a set of equivalent

sites on a fluorescence molecule, the equilibrium between freeand bound molecules is given by the Eq. (2) [11]:

lgðF0 � FÞ=F ¼ lgK þ n lg CQ ð2Þ

where K and n are the binding constant and the number of bindingsites, respectively, which in CD-ctDNA system are listed in Table 2.It can be seen that 1 CD molecule combine with 1 ctDNA molecule.

In order to elucidate the interaction of CD with ctDNA, thethermodynamic parameters were calculated from the van’t Hoffplots. If the enthalpy change (DH�) and the entropy change (DS�)do not vary significantly over the temperature range studied, thentheir value can be determined from the van’t Hoff equation:

lnK ¼ �DH�=RT þ DS

�=R ð3Þ

Fig. 2. The Stern–Volmer curves of CD as ctDNA were added at differenttemperature. Conditions: pH 7.4, CD: 1.0 � 10�5 mol L�1; from 1 to 10: ctDNA(g L�1): 0, 0.01, 0.025, 0.04, 0.05, 0.075, 0.09, 0.10, 0.125, 0.15.

Table 1The quenching constants.

T (K) Ksv (107 L mol s�1) Correlation coefficient (r)

283 7.12 0.9815298 7.69 0.9879303 8.60 0.9946

Conditions: pH 7.4, CD: 1.0 � 10�5 mol L�1.

Table 2The binding constants of CD with ctDNA and the thermodynamic parameters.

T (K) n K (106) DG (KJ mol�1) DH (KJ mol�1) DS (J mol�1 K�1)

283 1.11 479 �47.025 �123.77 �271.2298 0.96 33.9 �42.957303 0.79 1.82 �37.150

Fig. 3. The effect of NaH2PO4 on the fluorescence quenching extent. j, CD-ctDNA;N, CD-yRNA. Conditions: pH 7.4; CD: 1.0 � 10�5 mol L�1; ctDNA: 5.0 � 10�2 g L�1;yRNA: 5.0 � 10�2 g L�1.

F. Wang et al. / Journal of Molecular Structure 927 (2009) 1–6 3

In Eq. (3), K is the binding constant at the corresponding tempera-ture and R is the gas constant. The temperatures used were 283,298 and 303 K. The enthalpy change (DH�) and the entropy change(DS�) were calculated from the slope of the van’t Hoff relationship,and they are listed in Table 2. The free energy change (DG�) wasestimated from the following relationship:

DG�¼ DH

� � TDS�

ð4Þ

The negative values of free energy (G), seen in Table 2, supports theassertion that the binding process is spontaneous. The negative en-thalpy (H) and entropy (S) values of the interaction of CD and ctDNAindicate that the binding is mainly enthalpy-driven and the entropyis unfavorable for it. Therefore, vander Waals interactions andhydrogen bonds played major role in the reaction [12,13]. Hydrogenbonds are specific and directed. They are probably best identifiedthrough their negative enthalpy of formation.

So, it is inferred that CD combines with ctDNA through vanderWaals interactions and hydrogen bonds, which occurrs the fluores-cence of CD quenched.

4. Mode of interaction between CD and nucleic acids

There are three models about binding of small molecules to theDNA double helix: intercalative binding, groove binding and elec-trostatic binding. Intercalation is defined when a planar, heteroar-omatic moiety slides between the DNA base pairs and bindsperpendicular to the helix axis. Groove binding generally involvesdirect hydrogen-bonding or van der Waals interactions with the

nucleic acid bases in the deep major groove or the wide shallowminor groove of the nucleic acid helix. Electrostatic binding inter-actions between cationic species and the negatively charged DNAphosphate backbone usually occur along the exterior of the helix.

4.1. Effect of the phosphate concentration on the fluorescencequenching extent

It is possible that there are electrostatic binding between CDand nucleic acids because the cations of codeine phosphates andthe negative electric charge of the phosphate group in nucleicacids. When NaH2PO4 is added to the CD-nucleic acids system ifwhich is binding with electrostatic attraction, the phosphate groupof NaH2PO4 will hinder the combine between CD and the phos-phate backbone of nucleic acids. So, the addition of NaH2PO4 cer-tainly will weaken the quenching effect of nucleic acids to theCD fluorescence, and the fluorescence of CD-nucleic acid systemsmust be enhanced along with the increase of the NaH2PO4 concen-tration [14].

The Fig. 3 is show the effect of the phosphate concentration onthe fluorescence quenching extent in this system. It can be seenthat the fluorescence intensity of CD-ctDNA or CD-yRNA systemhas almost no obvious influence when NaH2PO4 is added into thesystem. That is to say, the phosphate group has no influence tothe quenching of nucleic acids to the fluorescence of CD. Therefore,it is assured that the electrostatic attraction is not the main bindingmode between nucleic acids and CD.

4.2. Fluorescence quenching experiment

Since groove binding exposes the bound molecules to the sol-vent surrounding the helix much more than does the intercalatedspecies, we chose to determine the accessibility of CD to aqueousanionic quenchers in the presence of ctDNA. If CD is intercalatedinto the helix stack, it should be protected from the anionicquencher, owing to the base pairs above and below the intercala-tor. However, groove binding should provide much less protectionfor the chromophore. Even when groove bound, the negativelycharged phosphate groups are expected to repel the anionicquenchers from the helix surface. In aqueous solutions, iodide an-ion quenches the fluorescence of CD very efficiently, so we used KIas quencher to determine the relative accessibilities of the free andbound CD.

Fig. 4 shows the fluorescence quenching extents of different KIconcentrations on the CD, CD-fsDNA and CD-yRNA systems under

Fig. 4. The fluorescence quenching. j, CD; d, CD-ctDNA; N, CD-yRNA. Conditions:pH 7.4; CD 1.0 � 10�5 mol L�1; ctDNA: 5.0 � 10�2 g L�1; yRNA: 5.0 � 10�2 g L�1.

4 F. Wang et al. / Journal of Molecular Structure 927 (2009) 1–6

experimental conditions. It can be seen that the quenching tenden-cies of three curves are basically identical, which indicate that thebinding of CD and nucleic acids is not in the mode of intercalativebinding.

Fig. 5. The effect of the ionic strength on the fluorescence quenching extent. d, CD-ctDNA; N, CD-yRNA. Conditions: pH 7.4; CD: 1.0 � 10�5 mol L�1;ctDNA:5.0 � 10�2 g L�1; yRNA:5.0 � 10�2 g L�1.

Fig. 6. The absorption spectra of nucleic acids (a, ctDNA; b, yRNA). Conditions: pH 7.4; ctD1 � 10�5, 3 � 10�5.

4.3. Effect of the ionic strength on the fluorescence quenching extent

According to the previous reports [14], the great ionic strengthcan loose the double helix structure of DNA, which will affect thecombine between the small molecular ligand and DNA. With theincrease of ionic strength, the combine between small molecular li-gand and DNA will be descended in the groove binding while it willnot affect that combination in the intercalation. So, the change ofthe ionic strength in the system can be thought as a means to dis-tinguish groove binding from intercalation.

The influence of ionic strength on the fluorescence intensity ofCD-nucleic acids systems were tested as shown in Fig. 5. Experi-ments indicate that with the increase of ionic strength, the fluores-cence quenching extent has decreased. So, it is also assumed thatthe intercalation of CD and nucleic acids in this system isimpossible.

Above all, our experimental results are consistent that the inter-action between CD and nucleic acids is sorted to the groovebinding.

4.4. Absorption spectra

In order to understand the interaction mechanism, it is impor-tant to observe the molecular absorption features of the system.Nucleic acid display absorption peak at 260 nm which comes fromthe strong absorption of purine and pyrimidine bases in nucleicacids. Fig. 6 shows the absorption spectra of nucleic acids in thepresence and absence of CD. It can be seen that the absorption at260 nm of yRNA or ctDNA decreases a little but the position dosenot change when CD added into yRNA or ctDNA. The enhancementof the absorption of nucleic acids at 260 nm indicates that theextension of the nucleic acid chain caused by the weak hydrogenbonds decrease in nucleic acids [15]. So, we believe that CD bindingon the grooves of the nucleic acid helix by hydrogen bonds whichmade nucleic acid helix strong.

The absorption spectra show that the absorbance of ctDNA de-creased in the presence of CD and is same to that of yRNA. So it isalso proved that there is no impossibility for CD intercalative toDNA. It is believed that there are benzene ring connected somegroups in the structure of CD, which destroyed the polar of themolecule. So, it is impeded for CD to intercalate in the base pairsreplaced of the water molecular. Therefore, it is assumed that thecodeine molecule is binding on the groove of the nucleic acid helix;nucleic acid provides a hydrophobic micro-environment for the

NA: 5.0 � 10�2 g L�1; yRNA: 5.0 � 10�2 g L�1; from 1 to 4: CD (mol L�1): 0, 5 � 10�6,

F. Wang et al. / Journal of Molecular Structure 927 (2009) 1–6 5

codeine molecule. So, the fluorescence of CD was observed a smallblue shift accompanying decreased.

5. Energy transfer from codeine to nucleic acids

Fig. 7 show that there is an overlap between the absorptionspectra of nucleic acids and the fluorescence emission spectrumof CD which indicates that there is intermolecular energy transferfrom CD to nucleic acids.

In order to know more details of this transfer, the energy trans-fer efficiency (Ea) and the interaction distance (r) between donorand acceptor can be evaluated using Förster theory. This is a non-destructive spectroscopic method that can monitor the proximityand relative angular orientation of fluorophores, the donor andacceptor can be entirely separate or attached to the same macro-molecule. A transfer of energy could take place through direct elec-tro dynamic interaction between the primarily excited moleculeand its neighbors [16]. According to this theory, the distance ofbinding between CD and nucleic acids could be calculated by theequation [17,18]:

Fig. 7. The overlap spectra. (1) Fluorescence spectrum of CD; (2) absorptionspectrum of ctDNA; (3) absorption spectrum of yRNA Condition: pH 7.4; CD:1.0 � 10�5 mol L�1; DNA: 1.0 � 10�5 mol L�1; RNA: 1.0 � 10�5 mol L�1.

Fig. 8. FRETeff varying the acceptor/donor ratio. d, Morphia; j, codeine. Condition:pH 7.4; DNA:1.0 � 10�5 mol L�1; CD (� 10�5 mol L�1): 0.2, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8;morphia (�10�5 mol L�1): 0.2, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8.

Ea ¼R6

0

R60 þ r6

ð5Þ

R6o ¼ 8:8� 10�25 � k2 � n�4 � /d � J ð6Þ

J ¼R1

0 FðkÞeðkÞk�4dkR1

0 FðkÞdkð7Þ

Ea ¼ 1� FF0

ð8Þ

where the energy transfer efficiency Ea, the critical transfer radiusR0 (cm) and the distance between the acceptor and donor r are cal-culated. In Eq. (6) k2 is a factor describing the relative orientation inspace of the transition dipoles of donor (CD) and acceptor (ctDNA oryRNA); ud is the fluorescence quantum yield of the donor (CD) inthe absence of acceptor (ctDNA or yRNA); n is the refractive indexof the solvent; In Eq. (7) J (cm6 M�1) is the spectral overlap(Fig. 8) integral between the emission spectrum of donor (CD) andthe absorption spectrum of acceptor (ctDNA or yRNA); F (k) is thefluorescence intensity of the fluorescent donor (CD) at wavelengthk and e (k) is the molar absorption coefficients of the acceptor(ctDNA or yRNA) at wavelength k. So, the energy transfer efficiencyis based on Eq. (8). Under these experimental conditions, usingk2 = 2/3, n = 1.336, ud = 0.079 (the fluorescence quantum yield ofCD in the system is determined based on the reference method withquinine sulfate as the criterion [19]), the spectral overlap integral Jbetween the emission spectrum of donor (CD) and the absorptionspectrum of acceptor (ctDNA or yRNA) from 290 to 500 nm, the val-ues of Ea, Ro and r are listed in Table 3.

In our experiments, it is found that the quenching extent ofyDNA on the CD fluorescence is stronger than that of ctDNA. FromTable 3, it can be seen that the distance between CD and yRNA is1.06 nm, which is closer than the distance between CD and ctDNA2.43 nm. Therefore, the interaction between codeine and yRNA isstronger than that between codeine and ctDNA.

In our previous works, it is found that nucleic acids can quenchthe fluorescence of morphia by forming the static complex [20].Morphia interacts with nucleic acids also in the mode of groovebinding using the oxygen atom of two hydroxyls in its molecularstructure through hydrogen bond and hydrophobic forces. The dif-ference of codeine and morphia structure is the –OCH3 group. So itis assumed that codeine interact with nucleic acid by –OCH3 com-bines with the groove of nucleic acid helix through hydrogen bondor van der Waals force, which cause the excited quenching of co-deine fluorescence.

Moreover, in order to discrimination the interaction betweenCD and nucleic acids randomly versus those form oligomers, thedependence of FRET (fluorescence resonance energy transfer) effi-ciency on acceptor/donor ratio at fixed surface density was testedshown as Fig. 8[21]. From Fig. 8, it can be seen that the FRET effi-ciency (FRETeff) reduces along with the relative concentration ratioof donor (CD)/acceptor (DNA). Here, the FRETeff is the ratio of donor(CD) emission in the presence of acceptor to that in absence ofacceptor, because the fluorescence intensity of acceptor (nucleicacids) is too weak to measure. Compared to morphina, the FRETeff

of codeine to DNA reduced rapidly. Theoretical considerations pre-dict that the combined number of donors and acceptors is heldconstant; FRETeff for random interactions will be independent ofacceptor/donor ratios above a certain threshold. In contrast, FRETeff

for oligomers is highly dependent on the relative donor concentra-

Table 3The efficiency of energy transfer (Ea) and the critical transfer radius (R0).

System Donor Acceptor E J 10�14 cm6 M�1) R0 (nm) r (nm)

CD-ctDNA CD ctDNA 0.87 9.76 3.35 2.43CD-yRNA CD yRNA 0.98 0.45 2.00 1.06

6 F. Wang et al. / Journal of Molecular Structure 927 (2009) 1–6

tion and reducing the relative concentration of donors will increaseFRET to saturation. It is considered that the interaction betweenDNA and morphina or codeine derives from the forming ofoligomer.

6. Conclusion

In this work, it is found that in the medium of pH 7.40 nucleicacids can quench the fluorescence of codeine and the interactionmechanism of this system is investigated. It is considered that co-deine interacts with nucleic acids in a mode of groove binding and–OCH3 of the codeine molecular combines with the groove of nu-cleic acids through hydrogen bond or van der Waalsforce.

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