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Available online 21 October 2011

Vitamin B12Lysozyme

eenhrouo4, 7

terms of a static quenching process at lower concentration of B12 (C /C < 5) and a combined quench-

were probed, and their binding afnities were determined under different pH conditions (pH 3.4, 7.4,and 9.0). The effect of B12 on the conformation of Lys was analyzed using UV, synchronous uorescence

sistingimport

contains six tryptophan (Trp) and three tyrosine (Tyr) residues[4]. Three of Trp residues are located at the substrate binding sites,two in the hydrophobic matrix box, while one is separated fromthe others [4,5]. Trp62 and Trp108 are the most dominant uoro-phores [6], both being located at the substrate binding sites.

Investigation of binding mode between drug and protein undervarious pH conditions, especially at lower pH values, would pro-

cause anemia resulting from less healthy red blood cells or prob-lems with memory and confusion.

Several reports have been published on the interactions of Lyswith small molecules, such as tetracycline analogs [10], farrerol[11], surfactant [12], and myricetin [13]. However, the interactionmechanism of B12 with Lys has been not studied, including theinuence of pH on the interaction between B12 and Lys. In thiswork, we have carried out the research on the effect of three differ-ent pHs and metal ions on the interaction between B12 and Lysemploying uorescence and absorbance. Corresponding author. Tel.: +86 379 65523821.

Journal of Molecular Structure 1007 (2012) 102112

Contents lists available at

ec

lseE-mail address: lyhxxjbm@126.com (B. Ji).extensive use as a model system to understand the underlyingprinciples of protein structure, function, dynamics, and foldingthrough theoretical and experimental studies [1,2]. High naturalabundance is also one of the reasons for choosing Lys as a modelprotein for studying proteinligand interaction. Another importantaspect of Lys is its ability to carry drug or biological activity sub-stances [3], and the effectiveness of them depends on their bindingability. Therefore, studies on the interaction between Lys and drugsor biological activity substances are of importance in view of real-izing disposition, transportation and metabolism of drugs or bio-logical activity substances as well as efcacy process. Lys

each domain of the protein occurs and the transition states ofthe protein may affect the binding afnities [5], which would di-rectly inuence the concentration of the drug in the blood, there-fore, affect its biological actions.

Vitamin B12 (cobalamin, Cbl) is a member of the tetrapyrrolic-derived macrocyclic family of compounds, one of the most com-plex cofactors in nature. Cyanocobalamin (B12, see Scheme 1) con-sists of a central cobalt ion held within a corrin ring. The cyanogroup is covalently bound only to the central cobalt [9].

B12 is needed to keep our blood healthy and is used by the bodyto make proteins. Having a diet that is too low in B12 over time canFluorescence quenchingBinding constantThree-dimensional uorescenceSynchronous uorescence

1. Introduction

Lys is a small globular protein, conidues with four disulde bonds. The0022-2860/$ - see front matter 2011 Elsevier B.V. Adoi:10.1016/j.molstruc.2011.10.028and three-dimensional uorescence under different pH conditions. These results indicate that the bindingof B12 to Lys causes apparent change in the secondary or tertiary structures of Lys. Furthermore, theeffect of Zn2+ on the binding constant of B12 with Lys under various pH conditions (pH 3.4, 7.4, and9.0) was also studied.

2011 Elsevier B.V. All rights reserved.

of 129 amino acid res-ance of Lys relies on its

vide much information for profoundly understanding the pharma-cological actions of the drug and the relationships of theirstructures and functions [7,8]. The ligandprotein interaction iseven more complex when pH-induced conformational changes ofKeywords:

B12 Lys

ing process at higher concentration of B12 (CB12/CLys > 5). The structural characteristics of B12 and LysInvestigation on the pH-dependent bindiby uorescence and absorbance

Daojin Li a, Yumin Yang b, Xinxiang Cao a, Chen Xu a,aCollege of Chemistry and Chemical Engineering, Luoyang Normal University, Luoyang 4b Life Science Department, Luoyang Normal University, Luoyang 471022, China

a r t i c l e i n f o

Article history:Received 27 July 2011Received in revised form 15 October 2011Accepted 15 October 2011

a b s t r a c t

The binding reaction betwgated by uorescence, syncuorescence. The intrinsicpH buffer solutions (pH 3.

Journal of Mol

journal homepage: www.ell rights reserved.of vitamin B12 and lysozyme

oming Ji a,22, China

vitamin B12 (B12, cyanocobalamin) and lysozyme (Lys) has been investi-nous uorescence, ultravioletvis (UV) absorbance, and three-dimensionalrescence of Lys was strongly quenched by the addition of B12 in different.4, and 9.0) and the spectroscopic observations are mainly rationalized in

SciVerse ScienceDirect

ular Structure

vier .com/locate /molstruc

2. Materials and methods

which the spectrum only shows the spectroscopic behavior ofTyr and Trp residues of Lys, respectively.

The three-dimensional uorescence spectra were performedunder the following conditions: the emission wavelength scanrange was recorded between 240 nm and 440 nm, the excitationwavelength scan range was recorded from 200 to 360 nm at10 nm increments. The number of scanning curves was 17, andthe excitation and emission bandwidths were 5 nm.

3. Results and discussion

3.1. Structure characteristics of Lys under different pH conditions

UV absorption spectra were chosen to observe the change ofprotein secondary or tertiary structure under different pH condi-

Scheme 1. Chemical structure of vitamin B12 (B12, cyanocobalamin).

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360

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ApH = 3.4

EX (n

m)

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360B

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EX (n

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240 260 280 300 320 340 360 380 400 420 440

240 260 280 300 320 340 360 380 400 420 440

D. Li et al. / Journal of Molecular Structure 1007 (2012) 102112 1032.1. Materials

Chicken egg white Lys (molecular weight (MW) = 14.6 kDa) waspurchased from Sigma (USA). B12 was of analytical grade and pur-chased from Sinopharm Chemical Reagent Co. Ltd., China. All otherreagents were of analytical grade. Double-distilled water was usedthroughout experiments. The pH of the phosphate buffer solution(20 mmol/L) was adjusted to 3.4, 7.4, and 9.0 respectively, by add-ing NaOH solution to phosphoric acid or NaH2PO4 solution, and theionic strength was controlled by adding NaCl to buffer solutions asrequired. A 2 mM water solution of B12 was used for all bindingexperiments.

2.2. Apparatus and methods

A 3 mL solution, containing appropriate concentration of Lys([Lys] = 4.0 106 mol/L, pH 3.4, 7.4, and 9.0), was titrated by suc-cessive additions of a 2 mM solution of B12. Titrations were donemanually by using trace syringes.

Fluorescence measurements were performed on an F-4500spectrouorophotometer (Hitachi, Japan) equipped with 1.0 cmquartz cells. The uorescence emission spectra were then mea-sured at 298 K and were recorded in a wavelength range of 290500 nm following an excitation at 280 nm, the excitation and emis-sion bandwidths were 5 nm.

UV absorption spectra were measured on a U-3010 UV spectro-photometer equipped a 1-cm cuvette at 298 K.

Synchronous uorescence spectra were obtained by scanningsimultaneously the excitation and emission monochromator. Thewavelength interval (Dk) is xed individually at 15 and 60 nm, at

3.0200 220 240 260 280 300 320 340

0.0

0.5

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2.5pH = 3.4

pH = 7.4

pH = 9.0

Abs

orba

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Wavelength (nm)Fig. 1. Inuence of pH on the UV absorption spectra of free Lys. [Lys] = 4.0 lM, at298 K; pH was 3.4, 7.4, and 9.0, respectively.

m)240 260 280 300 320 340 360 380 400 420 440200

220

240

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280

2

EX (n

EM (nm)EM (nm)

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320

340

360

1

CpH = 9.0Fig. 2. The three-dimensional uorescence contour map of free Lys under differentpH conditions: (A) 3.4, (B) 7.4, and (C) 9.0; [Lys] = 4 lM.

der

h sc

0?64

0?41

0?33

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0?1155 1819 682.6

0? 360/360 280/339 230/33794

0?79

0?

r StTable 1Three-dimensional uorescence spectral characteristics of Lys and LysB12 system un

pH System Rayleig

3.4 Lys Peak position (EX/EM) 240/24Relative intensity, F 422.7?

B12/Lys Peak position (EX/EM) 240/24(1:1) Relative intensity, F 364.2?B12/Lys Peak position (EX/EM) 240/24(2:1) Relative intensity, F 316.2?B12/Lys Peak position (EX/EM) 240/24(10:1) Relative intensity, F 89.48?

7.4 Lys Peak position (EX/EM) 240/24Relative intensity, F 523.8?

B12/Lys Peak position (EX/EM) 240/24(1:1) Relative intensity, F 469.6?B12/Lys Peak position (EX/EM) 240/24(2:1) Relative intensity, F 445.6?B12/Lys Peak position (EX/EM) 240/24

104 D. Li et al. / Journal of Moleculations. UV absorption spectra of Lys in the buffer solution with pH3.4, 7.4, and 9.0 were shown in Fig. 1. It can be seen that its inten-sity decreases gradually from pH 3.4 to 9.0, and an apparent blueshift of the maximum absorption wavelength from 199 nm (pH7.4) to 195 nm (pH 3.4) and an apparent red shift from 199 nm(pH 7.4) to 204 nm (pH 9.0) was observed. The literature [14]shows that the peak in the 200 nm region in the difference spectraof proteins is related to changes in the conformation of the peptidebackbone associated with the helixcoil transformation [15]. Theabove results suggest that the conformation of Lys molecule is al-tered when the pH is changed from simulating physiological (pH7.4) to acidic (pH 3.4) or basic (pH 9.0) conditions.

Three-dimensional uorescence spectra have become a popularuorescence analysis technique in recent years [16]. It is well-known that three-dimensional uorescence spectrum can providemore detailed information about the change in the conformation ofproteins. Fig. 2 presents the contours of three-dimensional uores-cence spectra under three different pH conditions (pH 3.4, 7.4, and9.0) and the corresponding data are shown in Table 1. In Fig. 2, two

(10:1) Relative intensity, F 113.0? 13

9.0 Lys Peak position (EX/EM) 240/240?Relative intensity, F 334.9? 53

B12/Lys Peak position (EX/EM) 240/240?(1:1) Relative intensity, F 317.0? 41B12/Lys Peak position (EX/EM) 240/240?(2:1) Relative intensity, F 248.0? 33B12/Lys Peak position (EX/EM) 240/240?(10:1) Relative intensity, F 112.1? 11

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Wavelength (nm)Fig. 3. Representative UV absorption spectra of B12 ranging from 190 to 600 nm inphosphate buffer solutions with pH 3.4 (1), 7.4 (2), and 9.0 (3), respectively;[B12] = 24 lM.typical uorescence peaks (peak 1, peak 2) could be easily ob-served. For peak 1, it mainly reveals the spectral characteristic ofTrp residues. The excitation wavelength of peak 2 is 230 nm, whichis related to the conformation of the peptide backbone. It can beobserved from Fig. 2 and Table 1 that the uorescence intensityof peak 1 reduced in the buffer solution from pH 7.4 to 3.4 or9.0, while the uorescence intensity of peak 2 reduced graduallywith increasing the pH of the buffer solution from 3.4 to 9.0. Inaddition, the results showed that an apparent red shift of the emis-sion band of peak 1 from 341 nm (pH 7.4) to 345 nm (pH 9.0) has

8.3 1553 535.7360/360 280/338 230/3364.0 1343 445.3360/360 280/335 230/335 280/3807.7 491.2 104.3 192.8

360/360 280/345 230/3472.4 1029 504.1360/360 280/345 230/3473.3 877.7 394.6360/360 280/342 230/3434.1 768.7 334.3360/360 280/339 230/337 280/3827.5 270 112.3 156.6three different pH values (3.4, 7.4, and 9.0).

attering peaks Peak 1 Peak 2 Peak 3

360/360 280/339 230/3421.3 1722 778.1360/360 280/338 230/3393.7 1528 672.8360/360 280/337 230/3365.0 1370 593.2360/360 280/334 230/331 280/382.76 551.1 130.1 214.1

360/360 280/341 230/341

ructure 1007 (2012) 102112been occurred. Contrarily, the uorescence emission band has a lit-tle blue shift from 341 nm (pH 7.4) to 339 nm (pH 3.4). The uores-cence emission band of peak 2 has an apparent red shift from341 nm (pH 7.4) to 347 nm (pH 9.0) and a slight red shift from341 nm (pH 7.4) to 342 nm (pH 3.4). This illustrated that the con-formation of Lys molecule was altered when the pH was changedfrom physiological condition (pH 7.4) to acidic condition (pH 3.4)and basic condition (pH 9.0). However, the changes of Lys tertiaryor secondary structures from simulating physiological condition(pH 7.4) to basic condition (pH 9.0) might be more signicant thanthat from simulating physiological condition (pH 7.4) to acidic con-dition (pH 3.4), which was also in accordance with the results ob-tained from UV. The alteration of the protein structures wouldfurther impact the protein binding ability with drugs.

3.2. Structure characteristics of B12 under different pH conditions

The wavelength, intensities, and shapes of B12 UV absorptioncan change with the surrounding medium such as pH. The UVabsorption spectra of B12 in different pH buffer solutions (pH3.4, 7.4, and 9.0) are shown in Fig. 3. It can be observed fromFig. 3, B12 has three main UV absorption peaks at about 203 nm(peak 1), 278 nm (peak 2), and 361 nm (peak 3) at pH 7.4. It wasinteresting to note that the UV absorption spectrum of B12 at pH7.4 was obviously different from that at pH 3.4, and its intensity in-creased from pH 7.4 to 3.4. This implied that the structural charac-teristic of B12 molecule was altered when pH was changed fromphysiological (pH 7.4) to acidic (pH 3.4) conditions. In contrast,no obvious differences in the UV absorption spectra of B12 were

observed in the buffer solution with pH 7.4 and 9.0, which indi-cated the similar structural characteristics under these conditions.B12 would be associated with H+ under acidic conditions. There-fore, B12 molecule existed as a mixture of cationic species and neu-tral state at pH 3.4 while the B12 molecule existed as its neutralstate at pH 7.4 and 9.0.

3.3. Fluorescence quenching studies of Lys by B12 under different pHconditions

The intrinsic uorescence spectra of Lys in the presence of a ser-ies of concentrations of B12 under various pH conditions were col-

lected with excitation wavelength 280 nm (Fig. 4). It can be seenthat the intensity of Lys decreased regularly with the increase inconcentration of B12 and a slight blue shift of the maximum emis-sion wavelength was observed in the studied B12 concentrationunder various pH conditions (pH 3.4, 7.4, and 9.0). Furthermore,it was interesting to note that the uorescence spectrum of Lyssplits from a single peak into two peaks. In fact, a new uorescenceemission band at about 380 nm emerged. It can be observed fromFig. 3 that one non-binding B12 molecule has quite strong absorp-tion around 360 nm, which overlaps completely with the uores-cence of Lys. Thus, free B12 molecule in mixture of B12 with Lyscan absorb emitted light around 360 nm resulting in a dip in theintensity of the overall measured emission of Lys at this wave-length. This implied that the broad Lys emission band appears toresolve into two quite resolved bands with a second peak around380 nm.

To elucidate further the quenching mechanism of Lys inducedby B12, the uorescence quenching data are analyzed on the basisof the SternVolmer equation (Eq. (1) [17,18] as follows:

F0F 1 kqs0Q 1 KSV Q 1

here F0 and F are the relative uorescence intensities in the absenceand presence of quencher, respectively, [Q] is the concentration ofquencher, KSV the SternVolmer dynamic quenching constant, kq

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D. Li et al. / Journal of Molecular Structure 1007 (2012) 102112 105300 350 400 4500

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Fig. 4. Fluorescence spectra of B12Lys systems under different pH conditions: (A)3.4, (B) 7.4, and (C) 9.0. (111) The concentrations of B12 are (lM): 0, 4, 8, 12, 16,20, 24, 28, 32, 36, and 40. [Lys] = 4 lM.0 5 10 15 20 25 30 35 40 45

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pH = 3.4 pH = 9.0

Fig. 5. Plots of F0/F for Lys against [Q] of B12 ranging from 4 to 40 lM underdifferent pH conditions; [Lys] = 4 lM. Straight lines in the inset are plots of F0/F forLys against [Q] of B12 ranging from 4 to 20 lM. j pH 7.4; d pH 3.4; N pH 9.0.

Table 2Fluorescence quenching for the B12Lys interactions, T = 298 K.

pH KSV (104 L /mol) kq (1013 L /mol/s) R3.4 4.02 0.80 0.99667.4 5.12 1.02 0.99889.0 5.71 1.14 0.9946

Table 3Binding constants and binding sites for the studied pHs at 298 K.

pH Method 1 Method 2

K (L/mol) n R K (L/mol) n R

3.4 5.34 104 1.03 0.9974 4.36 104 1.49 0.99127.4 8.08 104 1.05 0.9995 5.72 104 1.35 0.9950

9.0 1.93 105 1.12 0.9983 7.68 104 1.69 0.9805

R is the regression coefcient.

the bimolecular quenching rate constant, s0 the average bimolecu-lar life-time in the absence of quencher evaluated at about 5 ns [1921]. Fig. 5 shows the plots of F0/F for Lys versus [Q] of B12 rangingfrom 4 to 40 lM and 4 to 20 lM (the inset of Fig. 5) of B12 at pH 3.4,7.4, and 9.0, respectively. Plots in Fig. 5 show upward curvature to-wards the y-axis at higher [Q]. This observation suggests that com-bined quenching (both dynamic and static) was involved at higherconcentrations of B12 as discussed in the literatures [2224].

As it is known, a linear SternVolmer plot represents a singlequenching mechanism, either static or dynamic [22]. At lower con-centration (CB12/CLys < 5) of B12, linear ttings (the inset in Fig. 5)of the experimental data obtained to Eq. (1) at different pH valuesafford KSV and kq (Table 2). From the results listed in Table 2, it canbe seen that the kq obtained under different pH values is aboutthree orders higher than the limiting diffusion constant Kdif of

10 1 1

Qf was not known in the experiment, to obtain the K and n accord-ing to the Eq. (3), Qf was replaced by the Qt (the total concentrationof the ligand), which was known in the experiment. For the B12Lyssystem in the lower concentration range of B12 (CB12/CLys < 5), thevalues for K and n under different pH conditions can be derivedfrom the intercept and slope of plots of log (F0 F)/F versus log[Qt] based on Eq. (3) and presented in Table 3. Linear regressionequations (Eqs. (4)(6)) at pH 3.4, 7.4 and 9.0 are expressed asfollows:

logF0 F=F 4:7275 1:03 logQt 4

logF0 F=F 4:9075 1:05 log Qt 5and

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106 D. Li et al. / Journal of Molecular Structure 1007 (2012) 102112the biomolecule (Kdif = 2.0 10 L mol s ) [25], which illus-trated that the specic interaction between Lys and B12 has oc-curred and their uorescence quenching mainly arose from staticquenching by complex formation [23,24].

In fact, in many cases, uorophores can be quenched by bothcollision and complex formation with the same quencher. Conse-quently, the SternVolmer plot will exhibit an upward curve, beingconcave towards the y-axis at higher [Q] [25] (Fig 5). Accordingly,F0/F is related to [Q] by the following modied form (Eq. (2)) of theSternVolmer equation [23,24]:

F0F 1 KDQ 1 KSQ 1 KD KSQ KDKSQ 2 2

where KD and KS are the dynamic and static quenching constants,respectively. It is second order in [Q] and thus leads to upward cur-vy plots of F0/F versus [Q] at higher [Q] arising from a combinedquenching (both dynamic and static) process.

3.4. Effect of pH on the binding constants of the B12Lys complex

In order to explore the inuence of pH on the B12Lys interac-tion, the binding parameters of B12 to Lys was investigated atthree different pH values (pH 3.4, 7.4, and 9.0).

When small molecules bind independently to a set of equiva-lent sites on a macromolecule, the equilibrium between free andbound molecules is given by the following equation [26,27] (Meth-od 1):

logF0 F

F logK n logQf 3

where K and n are the binding constant and the number of bindingsites, respectively. Qf is the free concentration of the ligand. Because

0.00325 0.00330 0.00335 0.00340 0.00345 0.0035011.15

11.20

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ln K1/T

Fig. 6. Vant Hoff plot for the interaction of Lys with B12 at pH 7.4.200 220 240 260 280 300 320 340-0.20.00.20.40.60.81.01.21.4

1.61.8

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Fig. 7. UV absorption spectra of the B12Lys system in different pHs: (A) 3.4, (B)7.4, and (C) 9.0. [Lys] = 4 lM; the concentrations of B12 are (14): 0, 4, 16 and32 lM. Curve 5: [Lys] = 0, [B12] = 4 lM.

logF0 F=F 5:2846 1:12 logQt 6

respectively.In addition, the binding constants and binding sites at three dif-

ferent pH values also can be given by the following equation [27](Method 2):

F0=F KQtF0=F0 F nKPt6 7

where Qt and Pt are the total concentrations of the ligand and theprotein, respectively. The values for K and n under different pH con-ditions can be derived from the intercept and slope of plots of F0/Fversus [Qt] F0/(F0 F) based on Eq. (7) and listed in Table 3. Linearregression equations Eqs. (8)(10) at pH 3.4, 7.4 and 9.0 are ex-pressed as follows:

F0=F 4:36 104Qt F0=F0 f 0:26017 8

F0=F 5:72 104Qt F0=F0 F 0:30887 9

and

F0=F 7:68 104QtF0=F0 F 0:52018 10respectively.

It can be inferred from Table 3 that there is about one bindingsite under different pH conditions. The binding constants showthat there is a modest strong binding force between B12 and Lys.The binding constants obtained by Method 1 did not completelyagree with the results obtained by Method 2 under various pH con-ditions. Because the Method 1 is an approximate method, it is sup-posed that the concentration of the binding ligand is far less thanthe total concentration of B12. So the results calculated by Method1 were not as accurate as the ones calculated by the other method(Method 2). The binding constants of B12 to Lys calculated fromMethod 1 under three different pH conditions (pH 3.4, 7.4 and9.0) is a little greater than the ones calculated from Methods 2.However, from the results we found that the binding constants cal-culated by the Method 1 and 2 are in substantial agreement. Thus,Method 1 also can be applied to evaluate the binding parameters

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240 260 280 300 320 340Wavelength (nm)Fig. 8. Synchronous uorescence spectra of Lys with various amounts of B12 whe260 280 300 320

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for ligandprotein interactions. The smaller the binding afnity ofligand to protein is, the closer the values of the binding constantscalculated by both Method 1 and Methods 2 are.

In Table 3, the binding afnity of B12 to Lys in the buffer solu-tion with pH 3.4 was a little lower than that at simulating physio-logical condition (pH 7.4). This result may come from thealterations of Lys structure or structural change of B12 with thechange in pH. From UV and three-dimensional uorescence asmentioned above, it could be inferred that there was a slight struc-tural change of Lys from pH 7.4 to 3.4, and thereby binding abilityof drug to Lys may be impacted [28,29]. Moreover, from the UVabsorption spectra of B12 as shown in Fig. 3, B12 is very sensitiveto the pH, which would cause different concentration of proton-

3.5. Thermodynamic parameters and nature of binding forces

3.6. Conformation investigation of Lys induced by B12 under different

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0.8 pH = 9.0 60 nm 15 nmFig. 9. The quenching of synchronous uorescence of Lys by B12 when Dk = 60 nmand 15 nm at different pH values (3.4, 7.4, and 9.0).pH conditions

3.6.1. UV absorption studiesUV absorption measurement is a relatively simple method and

applicable to explore the structural change and to know the com-plex formation [33]. UV absorption spectra of Lys with variousamounts of B12 under different pH conditions were presented inFig. 7. It can be observed that the UV absorption spectrum of Lysshows a strong band with a maximum at about 200 nm and a weakband with a maximum at 280 nm under different pH values. Theabsorbance of B12Lys system decreased gradually with increasingconcentration of B12 and the peak at about 200 nm has an appar-ent red shift under various pH conditions, which indicate the spe-cic interactions between B12 and Lys. Meanwhile, the resultsTo obtain information about the type of noncovalent interac-tions between B12 and Lys at pH 7.40, thermodynamic parameterswere calculated. Intermolecular interacting forces between a smallmolecule and a biomacromolecule include hydrogen bond, van derWaals force, electrostatic and hydrophobic interactions. Thermo-dynamic parameters for a binding interaction can be used as a ma-jor evidence to learn the binding mode. If the enthalpy changes(DH0) do not vary signicantly over the temperature range studied,then its value can be determined from the following equation:

lnK DH0=RT DS0=R 11

where K is the binding constant at the corresponding temperatureand R the gas constant. The binding constants of B12 to Lys acquiredby Method 1 were calculated to be 8.86 104 L mol1,8.08 104 L mol1, 7.09 104 L mol1at 288 K, 298 K and 308 K,respectively. The enthalpy change DH0 and entropy change DS0

were obtained from the slope and intercept of the linear vant Hoffplot (Fig. 6) of lnK versus 1/T based on Eq. (11). The values of DH0

and DS0 for B12 binding to Lys are evaluated to be 8.20 kJ mol1and 63.3 J mol1 K1, respectively. The positive DS0 value is fre-quently regarded as a typical evidence for a hydrophobic interaction[30]. Furthermore, the negative DH0 value (8.20 kJ mol1) ob-served cannot be mainly attributed to electrostatic interactionssince for electrostatic interactions DH0 is very small, almost zero[31,32]. Therefore, the negative DH0 and positive DS0 values indi-cate that both hydrophobic and hydrogen bond interactions a vitalrole in the binding reaction between B12 and Lys.ation state. It has been investigated from above section that B12molecule existed as its neutral state at pH 7.4, while it existed asa mixture of neutral state and cationic species after the protonationof B12 at pH 3.4. Thus, the binding ability of B12 to Lys at lower pHvalue (pH 3.4) may be affected greatly.

In contrast, the binding ability of B12 to the protein in buffersolution of pH 9.0 was greater than that in a solution of pH 7.4,simulating physiological conditions. B12 molecule also existed asits neutral state at pH 9.0. From the UV absorption spectra of B12as shown in Fig. 3, it could be inferred that there was no obviousstructural change of B12 from pH 7.4 to 9.0. This illustrated thatB12 did not inuence the binding ability of B12 to Lys. Therefore,the increase of the binding afnities may come from the alterationsof Lys structure.

ructure 1007 (2012) 102112imply that the interaction of B12 with Lys at various pH values(pH 3.4, 7.4, and 9.0) may cause the secondary and tertiary changeof Lys.

Br St360A

D. Li et al. / Journal of Molecula3.6.2. Synchronous uorescence spectroscopic studiesTo further investigate the structural change of Lys upon the

addition of B12, we measured synchronous uorescence spectros-copy of Lys (Fig. 8) with various concentrations of B12 under differ-

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Fig. 10. The three-dimensional uorescence contour map of Lys at pH 7.4.

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Fig. 11. The three-dimensional uorescence contour map of Lys at pH 3.4.360

ructure 1007 (2012) 102112 109ent pH values, which is a very useful method to study themicroenvironment of amino acid residues by measuring the possi-ble shift in wavelength emission maximum kem [34,35], the shift inposition of emission maximum corresponding to the changes of

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the polarity around the chromophore molecule, i.e., the conforma-tion of Lys. As is known, synchronous uorescence spectra showTrp residues of Lys only at the wavelength interval (Dk) of 60 nmand Tyr residues of Lys only at Dk of 15 nm.

It was observed from Fig. 8 that emission maximum of Trp res-idues was blue shifted (3 nm) while the emission maximum ofTyr residues remained unchanged with increasing concentrationof B12 under three different pH values. It indicates that the inter-action of B12 with Lys may cause a signicant conformationalchange of Trp residues micro-regions while it does not affect theconformation of Tyr residues.

It also can be seen in Fig. 9 that the addition of B12 results instrong uorescence quenching of Trp residues of Lys, which de-creases to about 76.8% in uorescence intensity, while uorescencestrength of Tyr residues decreases to about 66.8% at pH 7.4, simu-lative physiological condition. However, uorescence strength ofTrp residues of Lys decreases to about 72.0% and 74.1% at pH 3.4and 9.0, and that of Tyr residues reduced to about 62.1% and57.3% at pH 3.4 and 9.0, respectively. The above results are a signthat B12 will be apt to bind to Trp residues of Lys at various pHvalues.

3.6.3. Three-dimensional uorescence spectroscopic studiesIn recent years, three-dimensional uorescence spectrum has

been getting more importance in protein conformational analysisand can provide more detailed information about the change ofthe conguration of proteins upon the binding of ligand. The con-

explanation is that B12Lys complex came into being after theaddition of B12, making the diameter of Lys decreased, which inturn resulted in the weakening of the scattering effect.

As referred to peak 1, we think that it reveals the spectral char-acteristic of tryptophan residues. The uorescence intensity of thepeak decreased gradually and the maximum emission wavelengthof the peak showed a blue shift following the addition of B12 undervarious pH conditions (pH 3.4, 7.4, and 9.0). This blue shift effectindicates that conformational changes have occurred in the Lysstructure as the polarity around the Trp residues decreases andthe hydrophobicity increases, which was consistent with the resultobtained from synchronous uorescence spectroscopy.

Besides peak 1, there is another weak uorescence peak 2. Andthe excitation wavelength of this peak is 230 nm, which is relatedto the conformation of the peptide backbone associated with thehelixcoil. Comparing with the UV absorbance spectra of Lys, thereis a strong absorption peak around 200 nm and this peak is mainlycaused by the transition of n? p of Lyss characteristic polypep-tide backbone structure C@O. It can be observed from Table 1 thatthe uorescence intensity of peak 2 decreased a lot and the emis-sion wavelength has an apparent blue shift after interacting withB12 at pH 3.4, 7.4, and 9.0. We can conclude that the polypeptidebackbone structures of Lys were changed.

Furthermore, it can be seen in Fig. 1012 and Table 1 that onenew three-dimensional uorescence peak 3 at emission wave-length of 380 nm emerged at higher concentrations of B12 underdifferent pH conditions, which was in accordance with the result

110 D. Li et al. / Journal of Molecular Structure 1007 (2012) 102112tours of three-dimensional uorescence spectra were displayed inFig. 1012 and the corresponding data were listed in Table 1 underdifferent pH conditions (pH 3.4, 7.4, and 9.0). In Fig. 1012, peak ais the Rayleigh scattering peak and two typical uorescence peaks(peak 1, peak 2) also could be easily observed in three-dimensionaluorescence contour map of Lys without or with B12 at pH 3.4, 7.4,and 9.0. From Fig. 1012 and Table 1, it can be seen that the uo-rescence intensity of peak a decreased with the increase of B12 un-der different pH conditions (pH 3.4, 7.4, and 9.0), the possible

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Fig. 12. The three-dimensional uorescence contour map of Lys at pH 9.0.obtained from uorescence as mentioned above. This has been ex-plained in the upper section.

3.7. Energy transfer between B12 and Lys under different pHconditions

Fluorescence resonance energy transfer (FRET) has becomewidely used in all applications of uorescence. It is an importanttechnique for investigating a variety of biological phenomena.

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[Lys] = 4 lM; the concentrations of B12 are (AD): 0, 4, 8 and 40 lM.

According to the Frster non-radiative resonance energy transfertheory [36,37], the efciency (E) of energy transfer is related tothe distance between Lys and B12 (r) and can be calculated byEq. (12):

E 1 FF0

R60

R60 r612

R0, the distance when the transfer efciency is 50%, is given by Eq.(13):

R60 8:8 1025K2N4UJ 13where N is the refractive index of medium, K2 the orientation factor,and U the quantum yield of donor. The spectral overlap integral (J)between the donor emission spectrum and the acceptor absorptionspectrum is evaluated by the following equation:

J R10 Fkekk4 dkR1

0 Fkdk14

where F(k) is the uorescence intensity of the donor in the wave-length range k to k + Dk and e(k) is the extinction coefcient ofthe acceptor at k.

The overlap of the absorption spectrum of B12 with the uores-cence emission spectrum of Lys at three different pH values is

shown in Fig. 13. In the present case, it has been reported thatK2 = 2/3, U = 0.15, and N = 1.4 [10,24]. Using Eqs. (12)(14) theparameters related to energy transfer from Lys to B12 in differentpH media are calculated and are presented in Table 4. From Table 4it is observed that the distance between the ligand and the proteinis markedly increased in an acidic medium (pH 3.4) whereas thereis no obvious difference in a basic medium (pH 9.0) compared withthat at the simulative physiological condition (pH 7.4). This will beexplained on the basis of effects of pH on the interaction betweenLys and B12.

3.8. Effect of Zn2+on the binding afnity of B12 to Lys at different pHs

Metal ions are vital to human body and play an essentiallystructural role on many proteins based on coordinate bonds. Thepresence of metal ions in plasma may affect interaction of drugsor biological activity substances with Lys. Effects of common metalions (e.g. Zn2+) on binding constant of B12Lys complex under dif-ferent pH conditions were investigated at 298 K.

The values of binding constants K acquired by Method 1 in thepresence of Zn2+ are listed in Table 5. It can be seen from Table 3and 5 that the presence of Zn2+ increased the binding constantsof B12Lys complex in buffer solution of pH 7.4 and 9.0. The higherbinding constants possibly result from this aspect as follow: Zn2+

induced a conformational change of Lys, which is more favorablefor B12 binding to Lys. Thus, the increase in binding constant of

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Table 4Energy transfer parameters for B12Lys interactions in different pH media.

pH R0 (nm) r (nm) E J (cm3 L mol1)

3.4 2.22 2.48 0.1298 5.169 10157.4 1.86 1.64 0.1591 1.809 10159.0 1.9 1.77 0.1519 2.064 1015

D. Li et al. / Journal of Molecular Structure 1007 (2012) 102112 1110

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ncepH = 9.0Fig. 13. Spectral overlap of Lys emission (curve a) with the absorption spectrum ofB12 (curve b) under different pH conditions: (A) 3.4, (B) 7.4, and (C) 9.0.Table 5Binding constants of the B12LysZn2+ complex calculated by Method 1.

pH System K (L/mol)

3.4 B12LysZn2+ 2.51 1047.4 B12LysZn2+ 2.46 1059.0 B12LysZn2+ 2.23 106

Fig. 14. Cartoon and surface representations of the structure of Lys shows thelargest pocket on the surface. The active site pocket with the catalytic residues(Glu35 and Asp52) in red and the other residues in the binding site (Gln57, Ile58,

Asn59, Trp62, Ile98, Ala107, Trp108) in light orange. (For interpretation of thereferences to color in this gure legend, the reader is referred to the web version ofthis article.)

B12Lys in presence of Zn2+ prolongs storage period of B12 inblood plasma and weakens its maximum effects.

However, the binding constant of B12Lys in the presence ofZn2+ in the buffer solution with the pH 3.4 decreased comparedwith that in the absence of Zn2+, which possibly results from thecompetition between B12 and Zn2+ binding to Lys. The resultshows that the presence of Zn2+ reduces the B12Lys binding, caus-ing B12 to be quickly cleared from the blood, which may lead to theneed for more doses of B12 to achieve the desired medicinal effect.

Moreover, it can be observed from Table 5 that in the presenceof metal ion these data show a decrease in the binding constantson decreasing the pH from 9.0 to 3.4. Thereby, it can be elicitedthat the binding reaction of B12 and the protein in body is sensitiveto the change in pH.

ang Normal University (No. 10001017) for nancial support of thiswork.

References

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112 D. Li et al. / Journal of Molecular Structure 1007 (2012) 1021123.9. Putative binding site of B12 on Lys

The catalytic residues, Glu35 and Asp52, lie in a cleft that is con-tiguous to the largest pocket (with a volume of 84 3 and molecu-lar surface area of 73 2) harboring the binding site (Fig. 14). Thislargest depression on the protein surface would allow a close ap-proach by B12 providing the maximum contact surface area. More-over, as the binding constant is modest strong (Table 3), B12 can bereplaced by the substrate. Therefore, Lys may still function at themain level in the presence of B12.

The active site of Lys contains two Trp residues that are impor-tant for substrate binding (Fig. 14). Our conjecture of this is thatthe B12 binding site is also supported by the uorescence quench-ing data (Fig. 4), as this position would bring B12 in close proximityto the two Trp residues.

4. Conclusion

In the paper, the main purpose of this research was to study thebinding properties between B12 and Lys due to the great impor-tance of the binding in pharmacology and biochemistry. The uo-rescence quenching mechanism and binding constants wereinvestigated at pH 3.4, 7.4, and 9.0. Effect of pH on the bindingafnities of B12 to Lys may result from the alterations of proteinstructure or the structural change of B12 caused by change ofpH. These investigations also may provide some important theo-retic information for the improvement of the metabolism and dis-tribution of B12 in life science, cast some light on the future studyof the interaction between derivatives of B12 and other proteinase.

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Pharmacol. 46 (1993) 17211731.[32] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 30963102.[33] S.Y. Bi, D.Q. Song, Y. Tian, X. Zhou, X. Liu, H.Q. Zhang, Spectrochim. Acta Part A

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915919.

Investigation on the pH-dependent binding of vitamin B12 and lysozyme by fluorescence and absorbance1 Introduction2 Materials and methods2.1 Materials2.2 Apparatus and methods

3 Results and discussion3.1 Structure characteristics of Lys under different pH conditions3.2 Structure characteristics of B12 under different pH conditions3.3 Fluorescence quenching studies of Lys by B12 under different pH conditions3.4 Effect of pH on the binding constants of the B12Lys complex3.5 Thermodynamic parameters and nature of binding forces3.6 Conformation investigation of Lys induced by B12 under different pH conditions3.6.1 UV absorption studies3.6.2 Synchronous fluorescence spectroscopic studies3.6.3 Three-dimensional fluorescence spectroscopic studies

3.7 Energy transfer between B12 and Lys under different pH conditions3.8 Effect of Zn2+on the binding affinity of B12 to Lys at different pHs3.9 Putative binding site of B12 on Lys

4 ConclusionAcknowledgementsReferences