Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 440–446

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journal homepage: www.elsevier .com/locate /saa

Influences of pH, urea and metal ions on the interaction of sinomeninewith Lysozyme by steady state fluorescence spectroscopy

http://dx.doi.org/10.1016/j.saa.2014.04.0541386-1425/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Tel.: +86 379 69819251.E-mail address: lyhxxjbm@126.com (D. Li).

Daojin Li, Tian Zhang, Baoming Ji ⇑College of Chemistry and Chemical Engineering, Luoyang Normal University, Luoyang 471022, China

h i g h l i g h t s

� The fluorescence of native anddenatured Lys was quenched bysinomenine at pH 7.4.� The effect of metal ions on the

binding constant of sinomenine withLys was studied.� The pH 7.4 is the optimal acidity in

the binding reaction.� Sinomenine was harder to bind to the

denatured Lys.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 February 2014Received in revised form 4 April 2014Accepted 7 April 2014Available online 19 April 2014

Keywords:SinomenineLysozymeFluorescence quenchingUreaThree-dimensional fluorescenceMetal ions

a b s t r a c t

The interaction between sinomenine and Lysozyme (Lys) in aqueous solution has been systemicallyinvestigated by fluorescence spectroscopic techniques at pH 7.4. The quenching rate constants and bind-ing constants calculated indicated the static quenching mechanism and medium binding force. The effectof sinomenine on the conformation of Lys was analyzed using synchronous fluorescence and three-dimensional (3D) fluorescence. In addition, influence of pH on the binding of sinomenine to Lys wasinvestigated and the binding ability of the drug to Lys deceased under other pH conditions (pH 9.0,3.5, and 1.9) as compared with that at pH 7.4. As compared with the binding ability of sinomenine tonative Lys, that of sinomenine to denatured Lys deceases dramatically. Furthermore, the effect of manymetal ions on the binding constant of sinomenine with Lys was investigated.

� 2014 Elsevier B.V. All rights reserved.

Introduction

The interaction of protein with drugs has attracted great inter-est among researchers since several decades [1–3]. Lysozyme (Lys),an antimicrobial proteinase, consists of 129 amino acid residueswith four disulfide bonds. The importance of Lys relies on its exten-sive use as a model system to understand the underlying principlesof protein structure, function, dynamics, and folding through theo-

retical and experimental studies [4,5]. High natural abundance isalso one of the reasons for choosing Lys as a model protein forstudying protein–ligand interaction. Another important aspect ofLys is its ability to carry drugs [6] and the effectiveness of themmainly depends on their binding ability. Lys contains six trypto-phan (Trp) and three tyrosine (Tyr) residues [7]. Three of Trp resi-dues are located at the substrate binding sites, two in thehydrophobic matrix box, while one is separated from the others[7,8]. Trp62 and Trp108 are the most dominant fluorophores [9],both being located at the substrate binding sites. Therefore, studieson the interaction between Lys and drugs are of importance in

D. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 440–446 441

view of realizing the understanding of disposition, transportationand metabolism of drugs as well as efficacy process.

In addition, investigation of binding mode between drug andprotein under various pH conditions, especially at lower pH values,would provide much information for profoundly understandingthe pharmacological actions of the drug and the relationships oftheir structures and functions [10,11]. The drug–protein interac-tion may be less complex when pH-induced conformationalchanges of each domain of the protein occurs [11], which woulddirectly influence the concentration of drug in the blood, therefore,affect its biological actions. Thus, it is necessary to study the inter-action of drug and protein such as Lys under various pH conditions.

Furthermore, in this state of denaturation of Lys, weaker drug–protein interaction can result in obvious increase of drug concentra-tion in the blood. Sometimes this effect may cause toxic poisoning oreven lead to death [12]. Therefore, studies on the binding of drugwith Lys in the presence of chemical denaturant (urea) can improveinterpretation of the metabolism and transporting process of drug,which is helpful to explain the relationship between the structureand the function of the protein.

Moreover, there are also some metal ions present in body,which can affect the binding of the drugs and protein. They couldparticipate in many biochemical processes. Some proteins usuallyact as sequestration agent of metal ions and have a variety of metalsites with different specificities and the phenomenon of Lys molec-ular conformational alteration caused by metal ions-Lys bindingcould be observed. So it is reasonable to ratiocinate that metal ionswould affect the interaction of medicine molecules with Lys, andthus it would influence the distribution, pharmacological property,and metabolism of medicine in body.

Owing to great importance on the binding study of drugs withproteins in pharmacy, pharmacology and biochemistry, our grouphas put many efforts in this direction in current years [13–24].The interaction of Lys and several small molecules, such as vitaminC [22], baicalein [15], benzocaine, [16] and B12 [13] have beeninvestigated in recent years. However, there is no report on thebinding of sinomenine to Lys, especially about effect of pH, ureaand metal ions on the interaction between sinomenine and Lys.

Sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-meth-ylmorphinan-6-one) (Fig. 1) is one of alkaloids extracted fromChinese medical plant, Sinomenium acutum [25–27]. It has beenwidely used to decrease some effects of rheumatoid arthritis (RA)and neuralgia because of excellent clinical anti-rheumatic andanti-inflammatory properties and little side effects [28–31]. Itsanti-rheumatic effects are thought to be primarily mediated viarelease of histamine [29], but other effects such as inhibition of

Fig. 1. Chemical structure of sinomenine.

prostaglandin, leukotriene and nitric oxide synthesis may also beinvolved [30]. However, little is known about their active princi-ples and even less about their mechanisms of action. This mayaffect the future application of this drug. Therefore, it is of greatimportance to explore the interaction of sinomenine and Lys undervarious conditions.

In this study, we first reported the binding studies of sinome-nine to Lys at pH 7.4, which presented the static fluorescencequenching mechanism and medium binding constant. In addition,influences of pH, urea and metal ions on the interaction of sinom-enine with Lys were systemically investigated by steady statefluorescence.

Materials and methods

Materials and preparation of solutions

Chicken egg white Lys (molecular weight (MW) = 14.6 kDa) waspurchased from Sigma (USA). Sinomenine was of analytical grade,and purchased from the National Institutes for Food and DrugControl, China. All other reagents were of analytical grade. Double-distilled water was used throughout experiments. The pHs of thephosphate buffer solution (20 mmol/L) were adjusted to 1.9, 3.5,7.4 and 9.0. The concentration of Lys in the buffer was preparedusing the molecular weight of 14.6 kDa, and the final concentrationwas checked by comparing the measured absorbance with thepublished value (optical absorbance at 279 nm) 0.531 (1 g/L)).The stock solution (2 � 10�3 mol/L) of sinomenine was preparedby dissolving appropriate amount of sinomenine in 10 ml anhy-drous methanol. For the determination of fluorescence quenching,the quenching rate constants, and binding constants, the concen-tration of Lys was 4 lM.

Apparatus and methods

Fluorescence measurements were performed on an F-4500spectrofluorophotometer (Hitachi, Japan) equipped with 1.0 cmquartz cells following an excitation at 280 nm. The widths of boththe excitation slit and the emission slit were adjusted at 5 nm. Thethree-dimensional fluorescence spectra were performed under thefollowing conditions: the emission wavelength scan range wasrecorded between 240 nm and 440 nm at 1 nm increments, andthe excitation wavelength scan range was recorded from 200 to360 nm at 5 nm increments. The number of scanning curves was34, and the excitation and emission bandwidths were 5 nm.

A 3 mL buffer solution, containing appropriate concentration ofLys ([Lys] = 4.0 lM, 1.9, 3.5, 7.4 and 9.0) was titrated by successiveadditions of a 2 mM solution of sinomenine. Titrations were donemanually by using trace syringes.

Results and discussion

Binding mechanisms of sinomenine with Lys

It is well known that fluorescence of Lys originates from trypto-phan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues. Actu-ally, the intrinsic fluorescence of Lys is mainly contributed by theTrp residue alone, because the Phe residue has a very low quantumyield and the fluorescence of Tyr is almost totally quenched whenit is ionized or near by an amino group, a carboxyl group, or a Trp[32]. That is, intrinsic fluorescence of Lys is due mainly to the Trpresidues.

Fig. 2 shows the fluorescence emission spectra of native Lyswith various amount of sinomenine following an excitation at280 nm which represents the maximum absorption of the protein.

300 350 400 450

0

400

800

1200

1600

2000

Fluo

resc

ence

Inte

nsity

Wavelength (nm)

1

9

Fig. 2. Fluorescence emission spectra of Lys in the presence of various concentra-tions of sinomenine at pH 7.4. (1–9) The concentrations of cinchonidine are (lM): 0,4, 8, 12, 16, 20, 24, 28, and 32. [Lys] = 4 lM. kex = 280 nm.

442 D. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 440–446

It could be observed that the fluorescence intensity decreases reg-ularly with the increase in sinomenine concentration, indicatingthat the fluorescence quenching mechanism may be rationalizedin terms of a static quenching process.

To confirm further the quenching mechanism induced by sin-omenine, fluorescence quenching data are analyzed with theStern–Volmer equation (Eq. (1)) [33–44]:

F0

F¼ 1þ kqs0½Q � ¼ 1þ KSV ½Q � ð1Þ

where F0 and F are the relative fluorescence intensities in theabsence and presence of quencher, respectively, [Q] is the concen-tration of quencher, kq the biomolecular quenching rate constant,s0 the average bimolecular life-time in the absence of quencherevaluated at about 5 ns [22,33] and KSV is the Stern–Volmerdynamic quenching constant, which was determined by linearregression of a plot of F0/F against [Q]. The solid line in Fig. 3 showsthe lines of best fit of the experimental data to the Stern–Volmerequation. The values for KSV and kq, are presented in Table 1. Thevalue of kq calculated is greater than the maximum dynamic colli-sional quenching constant (2.0 � 1010 L mol�1 s�1) of various kindsof quenchers with biopolymers [45,46]. The result implies that astatic quenching mechanism is operative in the present complex.

For the static quenching process, the equilibrium between freeand bound molecule is given by the equation (Eq. (2)) [47,48]:

1.0

1.2

1.4

1.6

F 0/F

[Q] (10-6M)

0 5 10 15 20 25 30 35

Fig. 3. Plots of F0/F for Lys against sinomenine concentration ranging from 4 to32 lM; [Lys] = 4 lM.

logðF0 � FÞ

F¼ log K þ n log½Q � ð2Þ

where K is the binding constant and n the number of binding sites.The values for K and n can be calculated by plotting log (F0 � F)/Fversus log [Q] based on Eq. (2) as shown in Fig. 4 and presentedin Table 1. The value of n indicates that one molecule of Lys com-bined with one molecule of the drug. The result, along with thequenching result, again indicates that sinomenine can bind to Lysvia medium binding force.

Analysis of the conformation of Lys upon addition of sinomenine

Synchronous fluorescence spectroscopic studies of LysSynchronous fluorescence spectroscopy is a very useful method

to study the microenvironment of amino acid residues by measur-ing the emission wavelength shift [49,50] and has several advanta-ges such as sensitivity, spectral simplification, spectral bandwidthreduction and avoiding different perturbing effects [51]. Vekshin[52] suggested a useful method to study the environment of aminoacid residues by measuring the possible shift in wavelength emis-sion, the shift in position of emission maximum corresponding tothe changes of the polarity around the chromophore molecule.

As is known, synchronous fluorescence spectra show Trp resi-dues of Lys only at the wavelength interval (4k) of 60 nm andTyr residues of Lys only at 4k of 15 nm. As such, Fig. 5A and B pre-sented the effect of the addition of sinomenine on the synchronousfluorescence spectra of Trp and Tyr residues in Lys, respectively. Itcan be seen from Fig. 5 that the maximum emission wavelengthkept the position at the investigated concentrations range regard-less of 4k = 15 nm or 4k = 60 nm. It implies that the interaction ofsinomenine with Lys does not significantly affect the polarityaround Trp and Tyr residues microregions. It is possible that thebinding of sinomenine to Lys does not causes apparent change inconformation of Lys.

Three-dimensional fluorescence spectroscopic studiesThree-dimensional fluorescence spectra have become a popular

fluorescence analysis technique in current years [53]. As wellknown, the three-dimensional fluorescence spectrum can providemore detailed information about the change of the configurationof proteins. In addition, the contour map can also provide a lot ofimportant information. Fig. 6 presents the three-dimensional fluo-rescence spectra and contour ones of Lys or sinomenine–Lys. Thecontour map displayed a bird’s eye view of the fluorescence spec-tra. Peak a is the Rayleigh scattering peak and two typical fluores-cence peaks (peak 1, peak 2) could be easily observed in isometricthree-dimensional projection or three-dimensional fluorescencecontour map of Lys with various amounts of sinomenine. FromFig. 6 it can be seen that the fluorescence intensity of peak aincreased with the increase of sinomenine. The possible explana-tion is that the formation of a sinomenine–Lys complex made thediameter of Lys increased, which in turn resulted in the enhance-ment of the scattering effect.

As referred to peak 1, we think that it mainly reveals the spec-tral characteristic of tryptophan and tyrosine residues. The reasonis that when Lys is excited at 280 nm, it mainly reveals the intrinsicfluorescence of tryptophan and tyrosine residues. The fluorescenceintensity of the peak decreased markedly and the maximum emis-sion wavelength of the peak was not changed following the addi-tion of sinomenine (Table 2). It implies that the addition ofsinomenine caused static quenching of Lys, but did not changethe polarity of this hydrophobic microenvironment (tryptophanand tyrosine residues) of Lys. Besides peak 1, there is a new weakfluorescence peak 2. And the excitation wavelength of this peak is230 nm, which is related to the conformation of the peptide

Table 1Fluorescence quenching constant and binding constant of sinomenine–Lys system under different pH conditions, T = 298 K.

Lys state KSV (�104 L/mol) kq (�1012 L/mol/s) R K (�104 L/mol) n R

pH = 7.4 Native 1.51 3.02 0.998 1.80 1.02 0.999Denatured 0.60 1.20 0.998 0.29 0.92 0.998

pH = 9.0 Native 1.20 2.40 0.999 0.15 0.80 0.998pH = 3.5 Native 1.19 2.38 0.999 0.74 0.95 0.999pH = 1.9 Native 1.21 2.42 0.999 0.47 0.91 0.999

-5.5 -5.0 -4.5-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

log

(F0-F

)/F

log [Q]

Fig. 4. Plot of log (F0 � F)/F for Lys versus log [Q]. [Lys] = 4 lM.

D. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 440–446 443

backbone associated with the helix–coil. The emission intensity ofsinomenine–Lys system decreased with the addition of sinome-nine, but the maximum emission wavelength of the peak wasnot changed. In addition, the maximum excitation wavelengthdoes not have an obvious shift. We can conclude that the interac-tion of sinomenine with Lys did not result in a conformationalchange of the protein.

Influence of pH on the interaction between sinomenine and Lys

In order to explore the influence of pH on the sinomenine–Lysinteraction, the protein fluorescence quenching data of Lys in thepresence of sinomenine was analyzed in buffer solutions underfour different pH conditions at an excitation wavelength of280 nm. Fig. 7 shows plots fitted to the Stern–Volmer equation.The values for KSV and kq, are presented in Table 1. It can be inferred

280 300 320

0

50

100

150

200

250

300

Fluo

resc

ence

Inte

nsity

Wavelength (nm)

1

5

A

Fig. 5. Synchronous fluorescence spectra of Lys with various amounts of sinomenine. (1–4k = 15 nm and (B) 4k = 60 nm.

that the fluorescence quenching extent at pH 7.4 is much greaterthan that at pH 1.9, 3.5, or 9.0. This may imply that the bindingability of sinomenine to Lys is stronger at pH 7.4 than that at theother pHs, which can be successfully confirmed by the furtherdetermination of binding constant (K) of sinomenine with Lysunder four different pH conditions (see Fig. 8). The values for Kat pH 7.4, 9.0, 3.5, and 1.9 are also presented in Table 1. This resultindicated that Lys indeed exhibited the strongest binding affinitytoward sinomenine at pH 7.4. This phenomenon probably origi-nates from two aspects, protein and drug. As is seen from Fig. 9,the maximum emission wavelengths of Lys in the absence of sin-omenine shift from 341 nm to 338 nm when pH 7.4 changes into3.5 or 1.9. In addition, the fluorescence intensity of Lys is greaterthan that at pH 1.9, 3.5, or 9.0. These results indicate that the influ-ence of pH on the secondary structure of Lys is relatively great, andthereby binding capacity of drug to Lys may be impacted [54,55].Moreover, the drug may be a little sensitive to the pH owing tothe presence of N in the drug, which would cause different concen-tration of ionization state. Thus, its binding ability to Lys may beaffected.

3.4. The interaction of urea-induced Lys with sinomenine

The denaturation of protein can be induced chemically by usingurea, SDS, or acetone, etc. [45]. As we know, 8 M urea can lead tothe complete denaturation of Lys [56]. To explore the interactionof denatured Lys caused by urea with sinomenine, we carried outthe research on the fluorescence of denatured Lys with variousamount of sinomenine. The calculated KSV and kq, were determinedby linear regression curve (Fig. 10) of F0/F for against [Q] and listedin Table 1. This result suggests that the fluorescence quenchingprocess of denatured Lys by sinomenine may also be governedby a static quenching mechanism arising from a complexformation.

240 260 280 300 320

0

400

800

1200

1600

2000

Fluo

resc

ence

Inte

nsity

Wavelength (nm)

1

5

B

5) The concentrations of sinomenine are (lM): 0, 8, 16, 24, and 32; [Lys] = 4 lM. (A)

240280

320360

400440

0

500

1000

1500

200

240280

320360

A (1) peak a

peak 2peak 1

Fluo

resc

ence

Inte

nsity

EX(nm

)

EM (nm)240 260 280 300 320 340 360 380 400 420 440

200

220

240

260

280

300

320

340

360

A (2)

peak a

1

2

EX (n

m)

EM (nm)

240280

320360

400440

0

500

1000

1500

2000

200

240280

320360

B (1) peak a

peak 2

peak 1

Fluo

resc

ence

Inte

nsity

EX(nm

)

EM (nm)

240 260 280 300 320 340 360 380 400 420 440200

220

240

260

280

300

320

340

360

B (2)

peak a

2

1

EX (n

m)

EM (nm)

Fig. 6. The three-dimensional fluorescence projections and three-dimensional fluorescence contour map of Lys before (A) and after (B) sinomenine addition. (A) [Lys] = 4 lMand (B) [Lys] = 4 lM; [sinomenine] = 16 lM.

Table 2Three-dimensional fluorescence spectral characteristics of Lys and Lys–sinomenine system at pH 7.4.

System Rayleigh scattering peaks Peak 1 Peak 2

Lys Peak position (EX/EM) 240/240 ? 360/360 280/341 230/341Relative intensity, F 509.7 ? 1089 1392 509.9

Sinomenine /Lys Peak position (EX/EM) 240/240 ? 360/360 280/340 230/341(4:1) Relative intensity, F 339.2 ? 1471 1094 358.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

pH = 7.4 pH = 9.0 pH = 3.5 pH = 1.9

F 0/F

[Q] (10-6 M)0 5 10 15 20 25 30 35

Fig. 7. Plots of F0/F for Lys against [Q] of sinomenine ranging from 4 to 32 lM underdifferent pH conditions; [Lys] = 4 lM.

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

pH = 7.4 pH = 9.0pH = 3.5pH = 1.9

log

(F0-F

)/F

log [Q]-5.4 -5.2 -5.0 -4.8 -4.6 -4.4

Fig. 8. Plots of log (F0 � F)/F for Lys versus log [Q] under different pH conditions;[Lys] = 4 lM.

444 D. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 440–446

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000Fl

uore

scen

ce In

tens

ity

Wavelength (nm)

a pH 7.4 b pH 3.5 c pH 1.9 d pH 9.0

a

bc

d

300 350 400 450 500

Fig. 9. Fluorescence emission spectra of Lys in different pHs: (a) 7.4, (b) 3.5, (c) 1.9,and (d) 9.0; [Lys] = 4.0 lM.

1.0

1.1

1.2

1.3

F 0/F

[Q] (10-6 M)0 5 10 15 20 25 30 35

Fig. 10. Plot of F0/F for denatured Lys against [Q] of sinomenine ranging from 4 to32 lM; [Lys] = 4 lM.

-5.5 -5.0 -4.5

-1.5

-1.0

-0.5

log

(F0-F

)/F

log [Q]

Fig. 11. Plot of log (F0 � F)/F versus log [Q] for denatured Lys/sinomenine system;[Lys] = 4 lM.

-5.4 -5.2 -5.0 -4.8 -4.6 -4.4

-2.0

-1.8

-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

Zn2+

Mg2+

Cu2+

Ca2+

K+

Fe3+

log

(F0-F

)/F

log [Q]

Fig. 12. Plots of log (F0 � F)/F for Lys versus log [Q] in the presence of metal ions.

Table 3Effects of metal ions on binding constants of sinomenine–Lys.

Metal ions K0 (�104 L/mol) n R K0/K

Zn2+ 0.156 0.83 0.991 0.087Mg2+ 301 1.56 0.999 167Cu2+ 0.641 0.95 0.995 0.356Ca2+ 10.5 1.25 0.997 5.83K+ 28.2 1.34 0.998 15.7Fe3+ 1.47 1.07 0.996 0.817

D. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 440–446 445

In order to investigate the effect of unfolding of Lys on the bind-ing ability of sinomenine to the protein, we evaluated the bindingconstant of sinomenine with denatured Lys by plotting log (F0/F)/Fversus log [Q] based on Eq. (2) as shown in Fig. 11 and the valuewas presented in Table 1. It can be observed that the binding

ability of Lys with sinomenine decreased in the denatured Lys. Itmay imply that studied drug in the hydrophobic pocket in thenative Lys was partially removed during denaturation [57].

Influence of metal ions on the binding affinity of sinomenine with Lys

Metal ions are vital to human body and play an essentiallystructural role on many proteins based on coordinate bonds. Thepresence of metal ions in body may affect interaction of drugs withLys. Effects of common metal ions on binding constant of sinome-nine–Lys complex at pH 7.4 were investigated at 298 K as shown inFig. 12 and the values of binding constants K were presented inTable 3. It can be seen from Table 3 that the presence of Mg2+,Ca2+ or K+ increased the binding constants of sinomenine–Lys com-plex. The higher binding constants possibly result from this aspectas follow: Mg2+, Ca2+ or K+ induced a conformational change of Lys,which is more favorable for sinomenine binding to Lys. Thus, theincrease in binding constant of sinomenine–Lys in presence ofthe above ions prolong storage period of sinomenine in body andweaken its maximum effects. However, in case of Zn2+, Cu2+, orFe3+ the binding constant of sinomenine–Lys decreased comparedwith the binding constant without these ions, which possiblyresults from the competition of metal ions and sinomenine bindingto Lys in the same binding site. The result shows that the presenceof common metal ions reduces the sinomenine–Lys binding, caus-ing sinomenine to be quickly cleared from the body, which maylead to the need for more doses of sinomenine to achieve thedesired medicinal effect.

Conclusion

In the paper, influences of pH, urea and metal ions on the inter-action of sinomenine with Lys were characterized by measuringthe fluorescence and the three-dimensional (3D) spectra. It canbe elicited that the binding reaction of sinomenine with theprotein in body is sensitive to the change in pH. The pH 7.4 is

446 D. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 440–446

the optimal acidity. In addition, the denatured Lys caused weakerbinding ability of sinomenine to the protein. The effect of manymetal ions on the binding constant of sinomenine with Lys wasstudied.

Fluorescence spectroscopy can be employed to explore theinteraction of drug with denatured protein. This study also canprovide important insight into the interactions of the physiologi-cally important protein with drugs. Besides, useful informationcan be also obtained about the effect of environment on the struc-tural features of the protein, which may be correlated to its phys-iologically activity. This study also will be helpful to structure-based drug design.

Acknowledgements

We are grateful to the National Natural Science Foundation ofChina (No. 21372112), and the Natural Science Foundation of Luoy-ang Normal University (No. 10001017) for financial support of thiswork.

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