Bd

Ta

b

a

ARRA

KDHIFC

1

bDnflap

aobtdoTcclhl

1d

Spectrochimica Acta Part A 93 (2012) 125– 130

Contents lists available at SciVerse ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

j ourna l ho me page: www.elsev ier .com/ locate /saa

inding of dihydromyricetin to human hemoglobin: Fluorescence and circularichroism studies

ingting Chena, Shajun Zhub, Yanfang Shanga, Cunwang Gea, Guoqing Jianga,∗

School of Chemistry and Chemical Engineering, Nantong University, Nantong, 226019, PR ChinaDepartment of General Surgery, Affiliated Hospital of Nantong University, Nantong, 226001, PR China

r t i c l e i n f o

rticle history:eceived 5 January 2012eceived in revised form 18 February 2012ccepted 27 February 2012

eywords:ihydromyricetin

a b s t r a c t

The binding reaction between dihydromyricetin (DMY) and human hemoglobin (HHb) was investi-gated systematically with various spectroscopic methods including fluorescence quenching technique,ultraviolet (UV)–vis absorption, synchronous fluorescence, circular dichroism (CD) spectroscopy. Theexperimental results showed that DMY effectively quenched the intrinsic fluorescence of HHb via staticquenching. DMY binds to HHb with a stoichiometry that varies from 0.972:1 to 0.906:1 as the temperatureincreases from 296 to 304 K. The DMY-HHb binding constants were determined to be K296 = 2.79 × 104

4 −1 −1

uman hemoglobin

nteractionluorescenceircular dichroism

and K304 = 1.18 × 10 L mol . The reaction is characterized by negative enthalpy (�H = −80.46 kJ mol )and negative entropy (�S = −186.72 kJ mol−1), indicating that the predominant forces in the DMY-HHbcomplex are van der Waals and hydrogen bonding forces. Based on the Förster’s theory of non-radiativeenergy transfer, the binding distance between DMY and the inner tryptophan residues of HHb was deter-mined to be 3.15 nm. Furthermore, the CD spectroscopy indicated the secondary structure of HHb is notchanged in the presence of DMY.

. Introduction

Ampelopsis grossedentata, a vine plant in South China, haseen used as a health beverage for thousands of years [1].ihydromyricetin (DMY, Fig. 1), the main bioactive compo-ent extracted from Ampelopsis grossedentata, is one kind ofavonoids which possessed many pharmacological functions, suchs reducing blood pressure, antibacterial, antioxidant, antitumorroperties, etc. [2–4].

Hemoglobin (Hb) is the iron-containing oxygen-transport met-lloprotein in the red blood cells. Hemoglobin in the blood carriesxygen from the respiratory organs (lungs or gills) to the rest of theody where it releases the oxygen for cell use, and collects the resul-ant carbon dioxide to bring it back to the respiratory organs to beispensed from the organism [5]. Hemoglobin exists as a tetramerf globin chains that is composed of two � and two � subunits.he � subunit contains 141 amino acid residues whereas � subunitontains 146 amino acid residues. There are two types of subunitontacts in a Hb molecular, i.e. �1�1 (or its �2�2 symmetry equiva-

ent) and �1�2 (or its �2�1 symmetry equivalent) [6]. Each subunitas one redox iron heme as its prosthetics group, and the heme is

ocated in the crevices at the exterior of the subunit [7].

∗ Corresponding author. Tel.: +86 513 85012853; fax: +86 513 85012851.E-mail address: guoqjiang@yahoo.com.cn (G. Jiang).

386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2012.02.109

© 2012 Elsevier B.V. All rights reserved.

Recently, the interactions between flavonoids and hemoglobinhave attracted great interest [8–14]. The distribution, metabolism,and efficacy of many drugs in vivo are closely related to their affini-ties toward hemoglobin [12]. Studies of the interaction of smallmolecules with hemoglobin are imperative and of fundamentalimportance. In this work, the binding interaction of DMY withhuman hemoglobin (HHb) was investigated to understand theirtransportation and distribution in vivo.

2. Materials and methods

2.1. Materials

Human hemoglobin (catalogue number H7379) was purchasedfrom Sigma–Aldrich Chemical Company (St. Louis, MO, USA) andused without further purification. DMY (≥98.0%) was obtained fromZelang Co. Ltd. (Nanjing, China). Tris(hydromethyl)aminomethane(Tris) was purchased from Acros (Geel, Belgium). The Tris–HClbuffer (0.05 M, pH 7.40) containing 0.10 M NaCl was selected tokeep the pH value and maintain the ionic strength of the solution.The 1.5 × 10−5 M stock solution of HHb was prepared with Tris–HClbuffer. The stock solution of dihydromyricetin (3.75 × 10−3 M)

was freshly prepared with methanol. The ultimate percentages ofmethanol in all solutions were below 1% (v/v). This low amount ofmethanol does not affect the structure of HHb. For circular dichro-ism (CD) spectra, the sodium phosphate buffer solution (20 mM, pH

126 T. Chen et al. / Spectrochimica Acta Part A 93 (2012) 125– 130

OHO

OH O

OH

OH

OH

OH

7Aws

2

tcoaEflewl0naU

2

2

Dtvb

2

s(oqcebirsusr

700600500400300

0.0

0.5

1.0

1.5

2.0

2502402302202102000

1

2

3

4

Ab

so

rban

ce

Wavelength / nm

a

b

c

b

c

a: [DMY]

b: [HHb]

c: [HHb-DMY]-[DMY]

Ab

so

rban

ce

Wavelength / nm

a

b

c

Fig. 2. UV–visible spectra of HHb in the presence of DMY for the wavelengthrange from 250 to 700 nm (T = 296 K, pH 7.4): (a) absorption spectrum of DMYonly; (b) absorption spectrum of HHb only; (c) difference absorption spectrumbetween DMY-HHb and DMY. CHHb = 5.0 �M, CDMY = 12.5 �M. Inset figure shows

Fig. 1. Structure of dihydromyricetin (DMY).

.4) was selected according to the requirement of the apparatus.ll other reagents and solvents were of analytical grade and usedithout further purification unless otherwise noted. All aqueous

olutions were prepared using newly double-distilled water.

.2. Apparatus

UV–vis absorption spectra were recorded on a UV-2450 spec-rophotometer (Shimadzu, Japan) equipped with 1.0 cm quartzells. Fluorescence spectroscopy measurements were performedn a RF-5301PC fluorometer (Shimadzu, Japan) equipped with

thermostatically controlled cell holder and 1.0 cm quartz cell.xcitation and emission slit width were 5 nm. The synchronousuorescence spectra were obtained by setting the excitation andmission wavelength interval (��) at 15 and 60 nm. CD spectraere recorded on a JASCO J-810 automatic recording spectropo-

arimeter (Tokyo, Japan) controlled by the Jasco software with a.1 cm quartz cell at room temperature, and the speed of scan-ing was 50 nm/min. The pH measurements were carried out on

PHS-3C exact digital pH meter (Cole-Parmer Instrument Co., IL,SA).

.3. Procedures

.3.1. UV–vis measurementsThe UV–vis measurements of HHb in the presence or absence of

MY were made in the range of 190–700 nm at 296 K. HHb concen-ration was fixed at 5 × 10−6 M while the DMY concentration wasaried from 0 to 1.75 × 10−5 M. As a control, the UV–vis spectra ofuffer titrated with DMY was also measured.

.3.2. Fluorescence quenching measurementsThe excitation wavelength of 280 nm was used, and the emission

pectra were recorded from 290 to 550 nm at two temperatures296 K and 304 K). Titration quenching experiments were carriedut as follows: a 3 ml HHb sample (5 × 10−6 M) was placed in auartz cell and was titrated by successive additions of DMY (con-entration from 0 to 1.75 × 10−5 M). The overall dilution did not

xceed 3.5%. All solutions were mixed thoroughly and kept 10 minefore measurements. Synchronous fluorescence spectra of HHb

n the absence and presence of different amount of DMY were alsoecorded by scanning the excitation and emission monochromatorimultaneously. The wavelength interval (��) was fixed individ-ally at 15 and 60 nm, at which the spectrum only showed thepectroscopic behavior of tyrosine and tryptophan residues of HHb,espectively.

their absorbance spectra for the wavelength range from 200 to 250 nm.

2.3.3. CD measurementsThe CD measurements of HHb in the presence or absence of

DMY were made in the range of 200–260 nm with three scansaveraged for each CD spectrum. HHb concentration was fixed at5 × 10−6 M while the molar ratio of DMY to HHb concentration was0:1, 1:1, 3:1 and 6:1. The buffer solution was selected as the blankand was automatically subtracted from each spectrum during scan-ning. Each sample was scanned for three times to average for a CDspectrum.

3. Results and discussion

3.1. UV–vis absorption spectra studies

UV–vis absorption measurement is a simple but efficaciousmethod to explore the structural changes of protein and to inves-tigate protein–ligand complex formation. In this study, we usedthe difference absorption spectroscopy to obtain spectra. UV–visabsorption spectra of HHb in the absence and presence of DMYwere obtained by subtracting the corresponding spectrum of DMYin the buffer from that of DMY-HHb complex. The UV–vis absorp-tion spectra of HHb without and with DMY are shown in Fig. 2. Aspresented in Fig. 2, it can be seen that HHb has three absorptionpeaks. The strong absorption peak at 220 nm reflects the frame-work conformation of the protein. The weak absorption peak at274 nm appears due to the aromatic amino acids (Trp, Tyr and Phe).The peak at 406 nm corresponds to the porphyrin-Soret band ofHHb [9]. In Fig. 2, with addition of DMY to HHb solution, both theabsorption peak at 220 nm and the peak at 274 nm increase, whichindicate that the addition of DMY leads to the loss of the HHb skele-ton structure, and promotes the exposure of aromatic ring aminoacids in the internal hydrophobic region. Furthermore, the Soretband of HHb decreases with no shift, suggesting that DMY can alsoaffect the structure of the heme group, but there is no direct interac-tion between DMY and the heme group. The UV–visible absorptionspectra of HHb (Fig. 2, curve b) and the difference absorption spec-tra between DMY-HHb complex and DMY (Fig. 2, curve c) could

not be superposed. These results indicated that there exists inter-action between DMY and HHb, and the ground state complex isformed.

T. Chen et al. / Spectrochimica Acta Part A 93 (2012) 125– 130 127

400380360340320300

0

50

100

150

200

250

300

350

400F

luo

rescen

ce In

ten

sit

y

Wavelength / nm

a

h

i

Fig. 3. Effect of DMY on fluorescence spectra of HHb (T = 296 K, pH 7.4). (a) 5.0 �MHHb; (b–h) 5.0 �M HHb in the presence of 2.5, 5, 7.5, 10.0, 12.5, 15, 17.5 �M DMY;(

3

itmttiattscapciTA3nrcHtlwo

3

waSttiuw

1.81.51.20.90.60.30.0

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7298 K

314 K

F0/F

[Q] ( 10-5 M)

dominated by a static quenching mechanism forming a DMY-HHbcomplex.

Table 1Stern–Volmer quenching constant (Ksv) and modified Stern–Volmer associationconstant (Ka) of the system of DMY-HHb at different temperatures.

T (K) 10−4 Ksv (L mol−1) 10−12 Kq (L mol−1 s−1) Ra SDb

296 3.83 3.83 0.9993 0.0097304 3.39 3.39 0.9980 0.0141

i) 17.5 �M DMY.

.2. Fluorescence quenching of HHb by DMY

For macromolecules, the fluorescence measurements can givenformation of the binding of small molecule substances to pro-ein at the molecular level, such as the binding mechanism, binding

ode, binding constants, intermolecular distances, etc. HHb con-ains three Trp residues in each �� dimer, for a total of six in theetramer: two �-14 Trp, two �-15 Trp, and two �-37 Trp [15]. Thentrinsic fluorescence of HHb primarily originates from �-37 Trpt the �1�2 interface, though it may contain some contribution byhe surface Trp residues, �-14 and �-15 Trp [16]. Like most pro-eins, the characteristic of the intrinsic fluorescence of HHb is veryensitive to its microenvironment. Some factors such as proteinonformational transitions, subunit association, substrate bindingnd denaturation, can result in the intrinsic fluorescence changes ofrotein [17]. Thus, the intrinsic fluorescence of proteins can provideonsiderable information about their structure and dynamics, ands often used to study the protein folding and association reactions.he effect of DMY on HHb fluorescence intensity is shown in Fig. 3.s seen in Fig. 3, HHb had a strong fluorescence emission band at32 nm by fixing the excitation wavelength at 280 nm. There waso fluorescence emission for DMY at the range measured. The fluo-escence intensity of HHb decreased regularly with increasing DMYoncentration, which is interpreted that DMY could interact withHb and quench its intrinsic fluorescence. A red shift (from 332

o 339 nm) was also observed for the maximum emission wave-ength, which suggests that the fluorescence chromophore of HHb

as placed in a more hydrophilic environment after the additionf DMY.

.3. The quenching mechanism

Since the intrinsic fluorescence of HHb can be quenched by DMY,e further explored the quenching mechanism. Quenching mech-

nisms are usually classified as either dynamic or static quenching.ince higher temperature results in larger diffusion coefficients,he dynamic quenching constants will increase with increasingemperature. In contrast, increased temperature is likely to resultn decreased stability of complexes, and thus leads to lower val-

es of the static quenching constants [18]. The quenching modeas primarily described as dynamic. In order to test this view,

Fig. 4. Stern–Volmer curves for the quenching of HHb fluorescence by DMY atdifferent temperatures (296 K and 304 K). CHHb = 5.0 �M; pH 7.4.

the fluorescence quenching data are analyzed by the well knownStern–Volmer Eq. (1) [19]

F0

F= 1 + KSV[Q ] = 1 + Kq�0[Q ] (1)

where F0 and F are the fluorescence intensities in the absenceand presence of the quencher, respectively. KSV, [Q], Kq, and�0 are the Stern–Volmer quenching constant, the concentrationof the quencher, the quenching rate constant of the biologicalmacromolecule and the fluorescence lifetime without quencher,respectively.

Fig. 4 displays the Stern–Volmer plots of the quenching ofHHb fluorescence by DMY at different temperatures. The plotshows that within the investigated concentrations, the resultsagree with the Stern–Volmer Eq. (1). Table 1 summarizes the cal-culated Ksv and Kq at each temperature studied. The Stern–Volmerquenching constant Ksv were 3.83 × 104 (296 K, R = 0.9993) and3.39 × 104 L mol−1 (304 K, R = 0.9980), respectively. The resultshows that the Stern–Volmer quenching constant Ksv is inverselycorrelated with temperature, which indicates that the probablequenching mechanism of the DMY-HHb binding reaction is ini-tiated by compound formation rather than by dynamic collision.In other hand, because the fluorescence lifetime of the biopoly-mer is 10−8 s [20], the quenching constants Kq for DMY werecalculated to be 3.83 × 1012 (296 K) and 3.39 × 1012 L mol−1 s−1

(304 K), respectively. According to the literatures, for dynamicquenching, the maximum scatter collision quenching constant ofvarious quenchers with the biopolymer is 2.0 × 1010 L mol−1 s−1

[21]. Considering that in our experiment the rate constant of theHHb quenching procedure initiated by DMY is much greater than2.0 × 1010 L mol−1 s−1, so it also proves that the overall quenching is

a R is the correlation coefficient.b SD is the standard deviation of the fit.

128 T. Chen et al. / Spectrochimica Acta Part A 93 (2012) 125– 130

Table 2Binding constant Kb and relative thermodynamic parameters of the system of DMY-HHb.

T (K) Ka (L mol−1) n Ra SDb �H (kJ mol−1) �S (J mol−1 K−1) �G (kJ mol−1)

296 2.79 × 104 0.972 0.9983 0.0184 −80.46 −186.72 −25.194 0214

3

itf

l

waccoswtvtib1stahcttwhdtw

Ffl

304 1.18 × 10 0.906 0.9974 0.

a R is the correlation coefficient.b SD is the standard deviation of the fit.

.4. Binding constants and number of binding sites

For the static quenching process, when small molecules bindndependently to a set of equivalent sites on a biomacromolecule,he equilibrium between free and bound molecules is given by theollowing equation [19,22]:

gF0 − F

F= lgKa + n lg[Q ] (2)

here F0 and F represent the fluorescence intensities of HHb in thebsence and presence of the DMY, respectively, Ka is the bindingonstant, n is the number of binding sites per HHb and [Q] is the con-entration of DMY. According to fluorescence spectra, the valuesf (F0 − F)/F can be obtained in each [Q]. Then, the linear regres-ion equation between the lg[(F0 − F)/F] values and lg[Q] valuesas obtained on Origin 7.5 software. The slope factor means n and

he intercept refers to lg Ka. Fig. 5 showed the plots of lg[(F0 − F)/F]ersus lg[Q] for the DMY-HHb system at different temperatures,he calculated binding constants (Ka) and the number of bind-ng sites (n) are presented in Table 2. The binding constants (Ka)etween DMY and HHb were 2.79 × 104 (296 K, R = 0.9983) and.18 × 104 L mol−1 (304 K, R = 0.9974), and the number of bindingites (n) was determined as 0.939 ± 0.033. These results illustratedhat there is a strong binding force between DMY and HHb, andlmost one molecular of DMY binds to one molecular of HHb withigh affinity. The correlation coefficients are larger than 0.997, indi-ating that the interaction between DMY and HHb agrees well withhe site-binding model underlying the Eq. (3). The value of Ka illus-rated that there is a strong binding force between DMY and HHb,hich implies that DMY can be tightly stored and carried by theemoglobin in the body. It was also found that the binding constant

ecreased with increase in temperature resulting in a reduction ofhe stability of DMY-HHb complex, which is in good agreementith the trend of KSV as mentioned above [23].

-4.6-4.8-5.0-5.2-5.4-5.6

-1.0

-0.8

-0.6

-0.4

-0.2

log

10 [

(F0

-F)/

F]

log10 [Q]

298 K

304 K

ig. 5. Plots of log10 (F0 − F)/F against log10[Q] for DMY quenching effect on HHbuorescence at different temperatures.

−23.70

3.5. Thermodynamic parameters and binding modes

The acting forces between small molecules and macromoleculesinclude hydrogen bonds, van der Waals forces, hydrophobic andelectrostatic interactions. Thermodynamic parameters, free energy(�G), standard enthalpy (�H) and standard entropy (�S) canprovide an insight into the binding mode [24]. Among these param-eters, �G reflects the possibly of reaction, �H and �S are the mainevidence to determine acting forces. When temperature varies in asmall range, the �H can be considered as a constant. Then throughthe binding constant Ka, thermodynamic parameters are evaluatedusing the following equations:

�G = −RT lnKa (3)

lnKa2

Ka1=

[1T1

− 1T2

]�H

R(4)

�G = �H − T�S (5)

where Ka2 and Ka1 are the binding constants at different tempera-ture and R is gas constant.

If �H < 0 and �S < 0, van der Waals’ interactions and hydro-gen bonds play a major role in the binding reaction. If �H > 0 and�S > 0, hydrophobic interactions are dominant. Electrostatic forcesare important when �H < 0 and �S > 0 [24]. The calculated valuesof Ka, �H, �S and �G for the binding interaction between DMYand HHb are listed in Table 2. As shown in Table 2, �H and �Sfor the binding reaction between DMY and HHb are found to be−80.46 kJ mol−1 and −186.72 J mol−1 K−1, respectively. The nega-tive �H and negative �S indicated that van der Waals interactionsand hydrogen bonds play a major role during the interaction. Thenegative sign of �G indicates that spontaneity of the binding pro-cess of DMY with HHb.

3.6. Energy transfer from HHb to DMY

According to the Förster non-radiative energy transfer theory[25], the energy transfer effect is related not only to the distancebetween the acceptor and donor, but also to the critical energytransfer distance (R0).

E = 1 − F

F0= R0

6

(R06 + r6)

(6)

where E is the energy transfer efficiency, R0 is the critical distancewhen the transfer efficiency is 50%, and r is the binding distancebetween donor and acceptor.

R60 = 8.79 × 10−25K2N−4˚J (7)

where K2 is the spatial orientation factor of the dipole, N is therefractive index of the medium, ̊ is the fluorescence quantumyield of the donor, and J is the overlap integral of the fluorescenceemission spectrum of the donor and the absorption spectrum of theacceptor, which could be calculated by Eq (8).∫ ∞

F(�)ε(�)�4d�

J = 0 ∫ ∞

0F(�)d�

(8)

Here F(�) is the fluorescence intensity of the fluorescence donorat wavelength � and ε(�) the molar absorptivity of the acceptor at

T. Chen et al. / Spectrochimica Acta Part A 93 (2012) 125– 130 129

4003803603403203000

50

100

150

200

250

300

350

400

0.0

0.1

0.2

0.3

0.4F

luo

rescen

ce In

ten

sit

y

Wavelength / nm

b

Ab

so

rban

ce

a

Fig. 6. The overlap of the UV–visible absorption of DMY with the fluorescence emis-st

wt

aJbdseItm

3

assmsiisarwFdrr3cawflafl

flatwr

340330320310300290280

0

20

40

60

80A: Δλ = 15 nm

Flu

ore

scen

ce In

ten

sit

y

Wavelength / nm

380360340320300

0

60

120

180

240

300

360

B: Δλ = 60 nm

Flu

ore

scen

ce In

ten

sit

y

Wavelength / nm

Fig. 7. Synchronous fluorescence spectra of interaction between HHb and DMY (A)at �� = 15 nm and (B) at �� = 60 nm. Concentration of HHb was 5.0 �M while con-centrations of DMY were 0, 2.5, 5, 7.5, 10.0, 12.5, 15, 17.5 �M from a to h (T = 296 K,pH 7.4).

1.81.51.20.90.60.30.0

0.6

0.7

0.8

0.9

1.0 Δλ = 15 nm

Δλ = 60 nm

[Q] /( 10-5 M)

F/F

0

ion spectrum of HHb: (a) the fluorescence spectrum of HHb, CHHb = 5.0 �M and (b)he UV–vis absorbance spectrum of DMY, CDMY = 5.0 �M, (T = 296 K, pH 7.4).

avelength �. The overlap of the absorption spectrum of DMY andhe fluorescence emission spectrum of HHb were shown in Fig. 6.

In the present case, N = 1.36, ̊ = 0.062 for Hb [26],ccording to the Eqs. (6)–(8), we could calculate that

= 1.63 × 10−14 cm3 L mol−1; E = 0.227; R0 = 2.36 nm; and theinding distance r = 3.15 nm. The average distances between aonor fluorophore and acceptor fluorophore are on the 2–8 nmcale [27], and 0.5R0 < r < 1.5R0 [28], which indicate that thenergy transfer from HHb to DMY occurs with high probability.n accordance with prediction by Förster’s non-radiative energyransfer theory, these results indicated again a static quenching

echanism between DMY and HHb.

.7. Synchronous fluorescence studies

Synchronous fluorescence spectroscopy can give informationbout the molecular environment in the vicinity of a chromophoreuch as tryptophan and tyrosine and it involve simultaneouscanning of the excitation and emission monochromators whileaintaining a constant wavelength interval between them. The

hift in the emission maximum (�em) reflects the changes of polar-ty around the chromophore molecule [29]. When the wavelengthnterval (��) between the excitation and emission wavelength istabilized at 15 or 60 nm, the synchronous fluorescence gives char-cteristic information of tyrosine residue or tryptophan residues,espectively [30]. The synchronous fluorescence spectra of HHbith various amounts of DMY are shown in Fig. 7. It can be seen from

ig. 7 that maximum emission wavelength of tyrosine residuesoes not have significant shift over the investigated concentrationange, which indicates that DMY has no effect on the microenvi-onment of tyrosine residues in HHb. In contrast, a red shift (from39 to 341 nm) of tryptophan residues was observed, which indi-ated that the hydrophobicity of the tryptophan residues decreasednd tryptophan residues buried in nonpolar hydrophobic cavitiesere moved to a more hydrophilic environment. Moreover, theuorescence intensity of HHb decreased regularly along with theddition of DMY, which further demonstrated the occurrence ofuorescence quenching in the binding process.

It has been also shown in Fig. 8 that addition of the DMY results in

uorescence quenching of tyrosine residues of HHb which decreasebout 37% in fluorescence intensity, and fluorescence strength ofryptophan residues decreases about 33%. The slope was higherhen �� was 15 nm indicating DMY was closer to the tyrosine

esidues than to the tryptophan residues.

Fig. 8. The quenching of HHb synchronous fluorescence by DMY. CHHb = 5.0 �M. (�)�� = 15 nm and (�) �� = 60 nm.

130 T. Chen et al. / Spectrochimica Acta

190 20 0 210 220 230 240 250-60

-30

0

30

60

90

120/

M-1

cm

-1

Wavelength / nm

HHb

DMY :HHb= 1

DMY :HHb= 3

DMY :HHb= 6

Fc(

3

oia22TnaCrosotts

4

usaqBdbttl

[

[

[[[

[

[[

[

[

[[[

[

[[

[

[[

[29] Y.Q. Wang, H.M. Zhang, G.C. Zhang, S.X. Liu, Q.H. Zhou, Z.H. Fei, Z.T. Liu, Int. J.

ig. 9. Far UV–CD spectra of HHb in the absence and presence of increasing con-entration of DMY in the range of 190–250 nm. CHHb = 5.0 �M, CDMY/CHHb = 0, 1,3,6T = 296 K, pH 7.4).

.8. Effect of DMY binding on the secondary structure of HHb

To ascertain the possible influence of DMY binding on the sec-ndary structure of HHb, CD measurement was also performedn the absence and presence of DMY. Consistent with the liter-ture, the CD spectrum for HHb (Fig. 9) monitored in the range50–190 nm shows the presence of two negative bands at 209 and22 nm, which are ascribed to the �-helix structure of protein [31].he negative peaks at 209 and 222 nm are both contributed to

→ �* transfer for the peptide bond of �-helix [32]. However, Fig. 9lso reveals that, for the DMY-HHb system, the appearance of theD spectra is exactly similar to that of HHb alone even though theatio of DMY to HHb is up to 6:1, which indicates that the structuref HHb after addition of DMY was also predominantly �-helix. Theuperimposed CD spectra of the HHb in the absence and presencef the DMY reveal that, at least in the experimental concentra-ions range, there is no perceptible secondary structural change ofhe HHb upon binding with the DMY, thus DMY hardly affects theecondary structure of HHb and can maintain protein stabilization.

. Conclusions

In this study, the interaction of DMY with HHb was investigatednder physiological conditions by spectroscopic methods includingteady-state fluorescence, synchronous fluorescence, UV–visiblebsorption, and circular dichroism spectroscopies. DMY effectivelyuenched the fluorescence of HHb by a static quenching process.ased on the results of binding capacity and calculated thermo-ynamic parameters, we concluded that DMY can spontaneously

ind with HHb through van der Waals and hydrogen forces. Fur-hermore, CD spectral result demonstrates that DMY hardly affectshe secondary structure of HHb and can maintain protein stabi-ization. The binding study of drugs to physiologically important

[[[

Part A 93 (2012) 125– 130

protein HHb is greatly important in pharmacy, pharmacology andbiochemistry, which may provide some references for the rationaluse of drugs in the clinic, and is helpful for clarifying the functionof HHb as drug storage.

Acknowledgments

The authors are grateful for the financial support from theNational Natural Science Foundation of China (nos. 21173122,61171015), the Natural Science Foundation of Jiangsu Province (no.BK2010281), and the Science and Technology Bureau of Nantong(no. K2009041).

References

[1] Y.S. Zhang, S. Que, X.W. Yang, B. Wang, L. Qiao, Y.Y. Zhao, Magn. Reson. Chem.45 (2007) 909–916.

[2] B.G. Liu, J.Q. Du, J. Zeng, C.G. Chen, S.Y. Niu, Eur. Food Res. Technol. 230 (2009)325–331.

[3] K.H. Lee, J.H. Kim, Biotechnol. Bioprocess Eng. 13 (2008) 274–278.[4] J. Ma, H. Yang, M.J. Basile, E.J. Kennelly, J. Agric. Food Chem. 52 (2004)

5873–5878.[5] F.W. Scheller, N. Bistolas, S.Q. Liu, M. Jänchen, M. Katterle, U. Wollenberger,

Adv. Colloid Interface Sci. 116 (2005) 111–120.[6] Y. Yuan, V. Simplaceanu, J.A. Lukin, C. Ho, J. Mol. Biol. 321 (2002) 863–878.[7] M.F. Chaplin, C. Bucke, Enzyme Technology, Cambridge University Press, Cam-

bridge, 1990.[8] T.T. Chen, S.J. Zhu, H. Cao, Y.F. Shang, M. Wang, G.Q. Jiang, Y.J. Shi, T.H. Lu,

Spectrochim. Acta A 78 (2011) 1295–1301.[9] Z.X. Chi, R.T. Liu, B.J. Yang, H. Zhang, J. Hazard. Mater. 180 (2010) 741–747.10] Y. Teng, R.T. Liu, S.F. Yan, X.R. Pan, P.J. Zhang, M.J. Wang, J. Fluoresc. 20 (2010)

381–387.11] S. Chaudhuri, B. Pahari, B. Sengupta, P.K. Sengupta, J. Photochem. Photobiol. B

98 (2010) 12–19.12] Y.Q. Wang, H.M. Zhang, Q.H. Zhou, Eur. J. Med. Chem. 44 (2009) 2100–2105.13] J.B. Xiao, J.L. Huo, F. Yang, X.Q. Chen, J. Agric. Food Chem. 59 (2011) 8484–8490.14] Z. Lu, Y.Y. Zhang, H. Liu, J.L. Yuan, Z.L. Zheng, G.L. Zuo, J. Fluoresc. 17 (2007)

580–587.15] Y.Q. Wang, H.M. Zhang, B.P. Tang, J. Photochem. Photobiol. B 100 (2010)

76–83.16] B. Alpert, D.M. Jameson, G. Weber, Photochem. Photobiol. 31 (1980) 1–4.17] A.S. Ladokhin, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, John

Wiley, Chichester, 2000, pp. 5762–5779.18] Y.Z. Zhang, B. Zhou, X.P. Zhang, P. Huang, C.H. Li, Y. Liu, J. Hazard. Mater. 163

(2009) 1345–1352.19] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Springer Sci-

ence + Business Media, New York, 2006.20] J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4161–4170.21] W.R. Ware, J. Phys. Chem. B 66 (1962) 455–458.22] J.B. Xiao, H. Cao, Y.F. Wang, K. Yamamoto, X.L. Wei, Mol. Nutr. Food Res. 54

(2010) S253–S260.23] F.L. Cui, L.X. Qin, G.S. Zhang, Q.F. Liu, X.J. Yao, B.L. Lei, J. Pharm. Biomed. Anal.

48 (2008) 1029–1036.24] D.P. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102.25] T. Förster, O. Sinanoglu, Modern Quantum Chemistry, Academic Press, New

York, 1996.26] A. Haouz, S. El Mohsni, C. Zentz, F. Merola, B. Alpert, Eur. J. Biochem. 264 (1999)

250–257.27] J.Y. Yang, W.Y. Yang, J. Am. Chem. Soc. 131 (2009) 11644–11645.28] B. Valeur, Molecular Fluorescence: Principles and Applications, Wiley Press,

New York, 2001.

Biol. Macromol. 41 (2007) 243–250.30] Y.Q. Wang, H.M. Zhang, R.H. Wang, Colloids Surf. B 65 (2008) 190–196.31] N.J. Greenfield, Nat. Protoc. 1 (2006) 2733–2741.32] S.M. Kelly, T.J. Jess, N.C. Price, Biochim. Biophys. Acta 1751 (2005) 119–139.