Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 349–356

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Spectroscopic and docking studies on the interaction between pyrrolidinium based ionic liquid and bovine serum albumin Meena Kumari a, Jitendra Kumar Maurya a, Upendra Kumar Singh a, Abbul Bashar Khan a, Maroof Ali b, Prashant Singh c, Rajan Patel a,⇑ a b c

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), New Delhi, India Department of Chemistry, Jamia Millia Islamia (A Central University), New Delhi, India Department of Chemistry, A. R. S. D. College, University of Delhi, Delhi, India

h i g h l i g h t s

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

 BMOP quenches the fluorescence

intensity of BSA and changes its conformation.  Quenching follows the dynamic mechanism.  BMOP are first used to investigate their effects on BSA.

a r t i c l e

i n f o

Article history: Received 1 November 2013 Received in revised form 26 December 2013 Accepted 5 January 2014 Available online 18 January 2014 Keywords: Ionic liquid Bovine serum albumin Fluorescence quenching Hydrophobic interaction Molecular docking

a b s t r a c t The interaction of synthesized ionic liquid, 1-butyl-1-methyl-2-oxopyrrolidinium bromide (BMOP) and bovine serum albumin (BSA) was investigated using UV–Vis, FT-IR, steady state and time resolved fluorescence measurements and docking studies. Steady state spectra revealed that BMOP strongly quenched the intrinsic fluorescence of BSA through dynamic quenching mechanism. The corresponding thermodynamic parameters; Gibbs free energy change (DG), entropy change (DS) and enthalpy change (DH) showed that the binding process was spontaneous and entropy driven. It is also indicated that hydrophobic forces play a key role in the binding of BMOP to BSA. The synchronous fluorescence spectroscopy reveals that the conformation of BSA changed in the presence of BMOP. The shift in amide I band of FT-IR spectrum of BSA suggested unfolding of the protein secondary structure upon the addition of BMOP. In addition, the molecular modeling study of BSA–BMOP system shows that BMOP binds with BSA at the interface between two sub domains IIA and IIIA, which is located just above the entrance of the binding pocket of IIA through hydrophobic and hydrogen bond interactions in which hydrophobic interaction are dominated. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Ionic liquids (ILs) are solvents that are often liquid at room temperature and composed of organic cation and appropriate anion. ⇑ Corresponding author. Tel.: +91 8860634100; fax: +91 11 26983409. E-mail addresses: [email protected], [email protected] (R. Patel). 1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2014.01.012

Their fascinating properties like negligible vapour pressure, nonflammability, high thermal stability, wide liquid range and large electrochemical window draws key interest of chemists [1,2]. ILs behaves as green surface active agents and can be superior over conventional surfactants in various chemical and biological applications. Till now no reliable rules exist so far that predicts the properties of any novel IL, even though some guidelines are

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zbrooming up, and quantitative structure–property relationships are being explored [3]. Currently, the research interest towards the biological application of ILs, grown up so rapidly because they provided exceptionally interesting solvent media for various biological processes viz. biocatalytic reaction [4], biosensor [5], protein separation, extraction and biopreservation [6,7]. The unusual solvating property and high thermal stability make them a novel protein stabilizing solvent. The spectroscopic analysis showed that [bmpy][NTf2] provide high thermal stability to the monellin [8]. Similarly, higher thermal stability of a-chymotrypsin, was observed in ammonium based ILs [9]. Beside these unique properties they also have prominent role in miscellaneous industrial applications, where high surface areas, modification of the inter-facial activity or stability of colloidal systems are required. Recently, the temperature dependent self-assembly of amphiphilic drug in [C8mim][Cl] have been reported [10]. Due to these interesting and useful applications of ILs herein, we examine the effect of synthesized pyrrolidinium based IL on BSA. The molecular interactions between them were studied systematically by fluorescence, time resolved fluorescence, UV–Vis, and FTIR spectroscopic techniques. In addition molecular docking was used to get better understanding of the interaction of BMOP with BSA. The most abundant proteins in plasma are serum albumins [11]. Serum albumin functions as the major transporter of non-esterified fatty acids and different drugs and metabolites to different tissues [12]. Serum albumin is synthesized in liver and exported as non-glycosylated protein. Bovine serum albumin (BSA) is one of most studied protein and used as a model in protein–ligand binding studies because of its abundance, low cost, easy purification [13,14], as well as of its structural homology with human serum albumin [15]. BSA is a globular protein of 582 amino acid residues [16]. The secondary structure of BSA consists mostly of a-helix, loops and disulphide bridges which unite to form a 3D heart shaped structure [17]. BSA consist of homologous domains I, II and III. Each domains are further subdivided into two subdomains A and B [18]. Among them, IIA and IIIA subdomains being hydrophobic in nature and serve as principle binding sites. BSA has two tryptophan residues, Trp-212 located in subdomain IIA, and Trp-134 in the subdomain IA. Tyrosine residues (Tyr-263) are also present in the subdomains IIA [19]. The spectroscopic techniques, such as UV–Vis spectroscopy [20], fluorescence spectroscopy [21], and Fourier transform infrared spectroscopy [22], are useful techniques to investigate the interaction between ligands and protein due to their non-destructive measurement of substances under physiological conditions with high sensitivity and rapidity [23]. To the best of our knowledge, the binding profile of BMOP with BSA has never been investigated. The aim of this study was to analyse the fluorescence quenching mechanism, binding properties and conformational changes induced by BMOP in BSA.

Experimental Materials BSA (96% purity) was purchased from Sigma Aldrich (Batch No. A2153) and was used without further purification. The BMOP was synthesized in the laboratory. All BSA solutions were prepared in phosphate buffer at pH 7.4. Doubly distilled water was used throughout the experiments. The stock solution of BSA was prepared in phosphate buffer (pH 7.4) and its concentration was determined from absorption spectroscopy. The stock concentration (20  106 M) was calculated by dividing absorbance at 280 nm by the molar extinction coefficients of the BSA e280 = 43,890 M1 cm1

[24]. The working concentration of BSA (5  106 M) was prepared by diluting the stock solution with phosphate buffer. Synthesis of BMOP The BMOP as shown in Fig. 1 was synthesized [25,26], by using the following method: in a round bottom flask (250 ml), N-Methyl pyrrolidone (10 mmol) in acetronitrile (50 ml) was taken and bromobutane (10 mmol) in acetronitrile (50 ml) was added to the above solution drop wise. After that the reaction mixture was refluxed for 3 h. Further, it was cooled at room temperature and solvent was evaporated under reduced pressure. It was further distilled under reduced pressure to afford the pure IL and then dried well and degassed at 60 °C for three day. It was well characterized by 1H NMR, 13C NMR, FTIR techniques and the purity of IL was checked by HPLC technique. FTIR (m = cm1 2935.05, 1705.87, 1400.25, 1365.89 and 865.49; NMR (d, CdCl3 1H NMR aliphatic proton at C3(2.49, 2H); aliphatic proton at C4(2.17, 2H); aliphatic proton at C5(3.19, 2H); aliphatic proton at C10 (3.05, 2H); aliphatic proton at C20 (1.98, 2H); aliphatic proton at C30 (1.56, 2H); aliphatic proton at C40 (1.11, 3H); aliphatic proton at C10 (2.95, 3H);13C NMR aliphatic carbon at 3, 4, 5, 10 , 20 , 30 , 40 and 100 are 35.6, 19.8, 50.2, 54.3, 30.7, 20.9, 15.6, 40.2 respectively and carbonyl carbon at 2 position is 184.9; HPLC purity of the synthesized IL is 98.1%. Procedures UV–Vis spectroscopy The UV–Vis spectra were measured using the Analytik Jena Specord-250 spectrophotometer (USA) using a 1.0 cm cell. The absorption spectra of BSA were recorded at different BMOP concentration at k280. Fluorescence spectroscopy The fluorescence spectra were recorded on a Cary Eclipse spectrofluorimeter (Varian, USA) equipped with a 150W xenon lamp in a 1 cm quartz cell and a thermostat water bath. The emission was measured from 290 nm to 450 nm with an excitation wavelength of 280 nm. Synchronous fluorescence spectra were acquired by the same spectroflourimeter. The difference between excitation and emission wavelength was kept constant (Dk = kem  kex). The Dk at 15 nm or 60 nm showed by synchronous fluorescence spectra gave characteristic information of Tyrosine (Tyr) residues or Tryptophan (Trp) residues, respectively, with the excitation and emission slit widths at 5 nm. Temperature was controlled during experiments using constant-temperature cell holder connected to constant-temperature water circulator (Varian, USA). Time-resolved fluorescence spectroscopy The time-resolved fluorescence measurements were performed at room temperature, using a single-photon counting spectrometer equipped with pulsed nanosecond LED excitation heads at 280 nm (Horiba, Jobin Yvon, IBH Ltd, Glasgow, UK). The fluorescence lifetime data were measured to 10,000 counts in the peak, unless otherwise indicated. The instrumental response function was

Br

N

O

Fig. 1. Schematic structure of the IL (BMOP) used in this work.

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recorded sequentially using a scattering solution and a time calibration of 114 ps/channel. Data were analysed using a sum of exponentials, employing a nonlinear least square reconvolution analysis from Horiba Jobin Yvon, IBH Ltd., of the form [27]:

i¼1

si

ð1Þ

The pre-exponential factors (ai) are shown normalized to 1 and the errors are taken as three standard deviations. The goodness of fit was judged in terms of both a chi-squared (v2) value and weighted residuals. Time-resolved fluorescence decays were analysed making use of the impulse response function (IBH DAS6 software). The average fluorescence lifetimes hsi for tri-exponential iterative fittings were calculated by using the following relation

P 2 ai s hsi ¼ P i ai si

0.30

Absorbance

  n X t f ðtÞ ¼ ai exp

0.35

i

0.25 0.20

a

0.15 0.10 0.05 0.00 240

FTIR spectroscopy Single pass attenuated total reflection Fourier transform infrared (ATR–FTIR) spectra were measured with a Specac Golden Gate diamond ATR sampler fitted to a Bruker Tensor 27 with an MCT detector. Approximately 4 ll of BSA solution was pipette onto a diamond window and a 100 scan interferogram was collected in single beam mode, with 2 cm1 resolution, from 1200 to 2000 cm1. Reference spectra were recorded under identical conditions with only the solvent media. Molecular docking The AutoDock 4.0 was employed to compute the possible binding mode of the BMOP with BSA. The 3-D structure of BMOP and BSA (PDB ID: 4F5S) were modeled on molecular modeling software pyMOL. The docking calculations were then performed using the Lamarckian genetic algorithm (LGA) for ligand conformational searching. Lamarckian genetic algorithm (LGA) implemented in the Autodock was used to estimate the possible binding conformations of BMOP. During the docking process, a maximum of 10 different conformations were considered for the BMOP. The conformer with the lowest binding free energy was used for further analysis. Results and discussion

Fluorescence spectra

300

320

340

380

400

protein conformation [32]. Shifts in kmax and fluorescence intensity which are mainly attributed to changes in the position of the Trp residues were used for studying the BSA–BMOP interaction. The lack of emission peak of Tyr at 300–310 nm was mainly attributed to the quenching of fluorescence by Trp due to efficient energy transfer from Tyr to Trp a result of internal quenching effect [33]. The BSA solution of fixed concentration of 5  106 M was excited at 280 nm, the maximum emission peak at 343 nm was observed. BMOP of different concentrations ranging from 4.15 to 29.20 mM was added to the BSA solution, which shows a decrease in fluorescence intensity regularly with its increasing concentration. Interestingly, a blue shift was observed from 343 to 335 nm in the fluorescence emission of BSA, with increasing concentrations of BMOP (Fig. 3), which indicates the change in conformation of BSA and shifts the Trp residues towards a more hydrophobic environment [34]. Fluorescence quenching decreases the fluorescence intensity of a fluorophore due to variety of molecular interactions namely excited state reactions, energy transfer, molecular rearrangements, ground state complex formation or collisional quenching [35– 36]. Usually fluorescence quenching is classified as dynamic quenching and static quenching. Dynamic quenching results from

600

Intensity (a.u.)

500

a 400 300

i

200 100 0 350

In case of macromolecules like protein, fluorescence measurements provide useful information regarding binding of the ligand to protein, such as the binding mechanism, binding site, and intermolecular distance [30,31]. There are three intrinsic fluorophore molecules tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) present in protein which provides the information about

360

Fig. 2. UV–Vis spectra of BSA in the absence (a) and presence (b-i) of BMOP (pH = 7.4). BSA = 5  106 M and BMOP = (b-i) 4.15–29.20 mM. The scanning was done at 280 nm with a time interval of 160 s per scan. 20 ll of 5% BMOP was added in 2 ml of BSA solution per spectrum.

UV–Vis spectra The UV–Vis measurements were used to explore the structural changes as well as to diagnose the complex formation between ligand and protein [28]. Fig. 2 shows the absorption spectra of BSA with increasing concentrations of BMOP in phosphate buffer at pH 7.4. An increase in absorbance of BSA (5  106 M) at 280 nm along with a blue shift in the spectra from 280 nm to 276 nm with increasing concentration of BMOP (from 4.15 to 29.20 mM) was observed. This indicates that BMOP binds with BSA and results in alteration in the conformation of BSA [29].

280

Wavelength (nm)

ð2Þ

where ai and si are the relative contribution and life time of different components to the total decay.

260

400

Wavelength (nm) Fig. 3. Fluorescence quenching spectra of BSA in the absence (a) and presence (b-i) of BMOP (pH = 7.4). BSA = 5  106 M and BMOP = (b-i) 4.15–29.20 mM at 298 K and pH 7.4. The spectra was taken at kex = 280 nm and kem = 290–450 nm with a time interval of 240 ± 5 s per scan. 20 ll of 5% BMOP was added in 2 ml of BSA solution per spectrum.

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the collision between the fluorophore and quencher, while static quenching results from the formation of a ground-state complex between the fluorophore and quencher. The two types of quenching mechanism, dynamic and static can be distinguished by the temperature dependent behaviour of their binding constants. In static quenching, the increase in temperature decreases the binding constant due to reduced stability of complex, on contrary the dynamic quenching, is marked by the increase in the binding constant with temperatures which leads to increase in collisions [37]. The quenching mechanism was analysed by the Stern–Volmer equation (Eq. (1)) [38]:

F0 ¼ 1 þ K sv ½IL ¼ 1 þ K q s0 ½IL F

ð3Þ

where F0 and F are the fluorescence intensities of BSA in the absence and presence of the BMOP, respectively. Ksv is the Stern–Volmer quenching constant, [IL] is the concentration of BMOP, Kq is the quenching rate constant of biomolecular reaction and s0 is the average life time of molecules in the absence of BMOP and its value is about 108 s [28]. The Ksv values are obtained from slope of the Stern–Volmer plot of BSA–BMOP system at different temperature (Fig. 4). The linearity of curve and increasing value of Ksv with temperature represents the involvement of dynamic quenching mechanism (Table 1) [39]. However, the values of quenching rate constant (Kq) for BSA–BMOP system are three orders of magnitude greater than maximum collision quenching rate constant of various kinds of biomolecule (2.0  1010 L mol1 s1) [40]. Therefore, the quenching mechanism was further conformed by analysing the fluorescence life time of intrinsic fluoprophore of BSA in presence of BMOP. Time resolved fluorescence spectra The fluorescence life time measurement is the most classic method to distinguish static and dynamic quenching [41]. Therefore, life time measurement was further used to conform the quenching mechanism for BSA–BMOP system. Life time measurements gather valuable information about conformational multifariousness of proteins. The folding/unfolding conditions prevailed largely due to the extent of solvent accessibility and several local configuration of proteins which determines the contribution of respective components [42]. Interaction of Trp residues in protein with the local environment and the solvent assimilates to provide the fluorescence lifetime [43]. The fluorescence lifetimes and statistical parameters were utilised to check the goodness of fit and the value conceivably agreed with the biexponential function. 298 K 303 K 308 K

2.2

2.0

Table 1 Stern–Volmer quenching constants of BSA–BMOP system at different temperatures (pH = 7.40, kex = 280 nm).

a

Temperature (K)

Ksv (105 L mol1)

Ra

298 303 308

3.45 3.94 4.33

0.9999 0.9996 0.9992

R correlation coefficient.

The measurement of fluorescence lifetime of BSA shows an interesting change in the mean lifetime of BSA (sm) as a function of ligand concentration. For static quenching, the sm value for BSA will not be disturbed during complex formation [21]. The lifetime components of BSA in presence of varying concentration of BMOP were lower compared to values of BSA in buffer. Table 2 shows the mean lifetime of BSA (sm) decreases from 6.05 ns to 5.13 ns with increasing concentration of BMOP. Therefore, the results clearly confirmed that the dynamic quenching was dominant in this reaction process. Similar results were obtained in case of BSA interacting with histidine-capped Au nanoclusters [44]. Binding constant and number of binding sites The fluorescence quenching due to binding of BMOP with BSA was further used to determine the binding parameters such as K and n. The value of K and n were calculated from the following equation [21,45]:

log

F0  F ¼ log K þ n log½IL F

ð4Þ

where K and n are the binding constant and number of binding sites, for BSA–BMOP complex, respectively. The plot of log F0  F/F versus log [IL] at three different temperatures gives a straight line shown in Fig. 5. The intercept of the curve is represented by K whereas the slope of such curve is equal to n. The values of the binding constant (K) and the number of binding sites (n) are listed in Table 3. The value of regression coefficient is also shown which is nearly equal to one. The value of n nearly equals to 1, which indicated that there is one binding site in BSA for BMOP. Thermodynamic parameters and nature of the binding forces The forces acting between biomolecule and the quencher primarily consist of hydrophobic forces, electrostatic interactions, van der Waals interactions, hydrogen bond and electrostatic forces [46]. The thermodynamic parameters, enthalpy change (DH), entropy change (DS) and free energy change (DG) were calculated to confirm the binding modes of BMOP with BSA. The values of DH and DS for BSA–BMOP system were obtained from the slope and intercept of the linear vant’t Hoff plot (Fig. 6) of ln K versus 1/T based on Eq. (5) [43]:

F0 /F

1.8

ln K ¼  1.6

DH DS þ RT R

ð5Þ

where K is the binding constant analogous to the Stern–Volmer quenching constants KSV at the corresponding temperature, R is the gas constant and T is the experimental temperature. The free energy change (DG) is estimated from the following relationship:

1.4

1.2

DG ¼ DH  T DS 1.0 0

5

10

15

20

25

30

[IL] (mM) Fig. 4. The Stern–Volmer plots of BSA quenched by BMOP at different temperatures (298, 303 and 308 K).

ð6Þ

Table 4, summarized the value of DG, DH and DS. The negative value of DG revealed that the interaction process was spontaneous. The positive values of DS and DH was evidence of hydrophobic interaction and a feeble hydrogen bond formation between BSA and BMOP, respectively [21,45].

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M. Kumari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 349–356 Table 2 Fluorescence decay of BSA at different concentrations of BMOP (pH = 7.40). Concentration (103 mol L1)

s1 (ns)

s2 (ns)

a1

a2

v2

sm

0.00 4.15 8.14 11.98 15.68 19.24 22.68 26.00

2.96 2.21 2.18 2.00 2.17 2.13 2.01 2.22

6.67 6.35 6.31 6.15 6.17 6.10 6.00 6.08

16.57 17.27 18.68 17.11 20.28 20.48 20.70 24.53

83.43 82.73 81.32 82.89 79.72 79.52 79.30 75.47

1.0444 1.2143 1.2477 1.2877 1.2207 1.2490 1.3242 1.4801

6.05 5.63 5.53 5.43 5.35 5.28 5.17 5.13

Synchronous fluorescence spectra

0.2

298 K 303 K 308 K

0.0

log F0 -F/F

-0.2

-0.4

-0.6

-0.8

-1.0 0.6

0.8

1.0

1.2

1.4

1.6

log [IL] (mM) Fig. 5. The plots of log (F0  F)/F versus log [IL] for quenching of BSA by BMOP at different temperatures.

Table 3 The binding constant and binding site of BSA and BMOP interaction at three temperatures. 5

T (K)

K (10 L mol

298 303 308

3.328 5.254 7.350

1

)

n

R

1.00 0.90 0.82

0.9997 0.9972 0.9978

13.00

12.95

ln K sv

12.90

12.85

12.80

12.75

0.00324

Synchronous fluorescence is a sensitive technique to explore the change in molecular environment of fluorophore residues due to various advantages such as its spectral bandwidth reduction, spectral simplification and avoiding different perturbing effect [47]. The maximum emission wavelength of fluorophore is related to the environmental polarity and shift in its position denote the changes of polarity around these fluorophore molecules. Synchronous spectra offer the characteristic information of Tyr and Trp residues when the Dk fixed between excitation and emission wavelength at 15 nm and 60 nm respectively [21]. Fig. 7A and B shows the synchronous fluorescence spectra of BSA with varying concentration of BMOP. The spectra were recorded with Dk = 15 nm and 60 nm with addition of BMOP. The results shows the presence of BMOP leads to fluorescence quenching of BSA, while no change in maximum emission wavelength were reported. A substantial quenching is recorded when Dk of 60 nm. This reveals that the BMOP have more quenching effect on Trp residue than Tyr residue in the interaction system. Similar result was also observed for imidazolium IL-BSA system by Liu et al. [48]. FT-IR characterization FT-IR technique is an important technique to monitor the conformational changes in the protein molecules [49]. Protein exhibits number of amide bands in infrared spectrum sensitive to secondary structure [50]. The amide I band at approximately 1600– 1700 cm1 is sensitive to change in protein conformation due to the C@O stretching vibration having significantly higher signal intensity. The amide II band occurs nearly 1548 cm1 due to CAN stretch coupled with NAH bending. Hydrogen bonding and the coupling between transition dipoles are the most important factors governing conformational sensitivity of the amide bands [51]. The FT-IR spectra of BSA, a peak at 1643 cm1 were observed from Fig. 8. Quiet interestingly, from Fig. 8; a decrease in the intensity of amide bands with a small shift in the amide I band was observed (from 1643 cm1 to 1640 cm1) in presence of BMOP (22 mM). These changes in infrared spectra of BSA confirmed the change in conformation of BSA on the addition of BMOP. Fig. 9a and b reveals a quantitative analysis of the protein secondary structure of BSA before and after the interaction with BMOP in the phosphate buffer, respectively. The FTIR spectra were smoothed, and their baselines were corrected automatically. The peak positions in spectral region 1700–1600 cm1 of amide I bands

Table 4 Thermodynamic parameters of the binding of BSA with BMOP. 0.00326

0.00328

0.00330

0.00332

0.00334

0.00336

1/T (K) Fig. 6. Vant’s-Hoff plot for the interaction of BSA and BMOP at different temperatures (pH = 7.4).

T (K)

DH (kJ mol1)

DS (J mol1 K1)

DG (kJ mol1)

R

298 303 308

17.22

163.92

31.62 32.44 33.26

0.9964

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M. Kumari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 349–356 200

(A)

40

180

(B) a

160

Intensity (a.u.)

Intensity (a.u.)

a

30

i 20

10

140 120

i

100 80 60 40 20

0

0 260

280

300

320

Wavelength (nm)

260

280

300

320

Wavelength (nm)

Fig. 7. Synchronous fluorescence spectra of BSA (5  106 M) in absence (a) and presence of various concentration (b-i = 4.15–29.20 mM) of BMOP (A) Dk = 15 nm; (B) Dk = 60 nm, (T = 298 K, pH = 7.4).

50 BSA-BMOP BSA

Transmitance

40

30

20

[52]. Upon BMOP interaction, there was a major decrease in a-helix from 55% to 49%, also in random coil from 27% to 18% and a minor decrease in b-sheet from 9% to 6% but an increase in b-antiparallel from 3% to 9% as well as major increase in b-Turn from 8% to 24%. These results again show the decrease in intensity of amide 1 band on the addition of BMOP as discussed above. In addition, the decrease in a-helix structure and increase in b-Turn and b-antiparallel structure suggested conformational changes in BSA induce by BOMP.

10 1643

0 1800

Molecular modeling

1640

1700

1600

1500

1400

1300

-1

Wavenumber (cm ) Fig. 8. FT-IR spectra of BSA in absence and presence of BMOP. BSA = 5  106 M and BMOP = 22 mM at 298 K and pH = 7.4.

are a-helix (1660–1650 cm1), b-sheet (1638–1610 cm1), turn (1680–1660 cm1), random coil (1648–1638 cm1), and b-antiparallel (1692–1680 cm1) respectively. The major peaks for protein and the complexes in the region mentioned above were resolved. The above spectral region was deconvoluted were adjusted and the area measured with the Gaussian function fitting curves. The area of all component bands assigned to a given conformation were then summed up and divided by the total area. For all the fittings, the square of the correlation coefficient (R2) is 0.9999 for BSA and 0.9998 BSA–BMOP systems. The spectra of free BSA obtained were found to be in consent with the reported data after curve fitting

Molecular docking technique has been employed to understand the different binding modes of BSA–BMOP interaction. The 3D structure of BSA was obtained from Proteins Data Bank. The possible conformations of the BSA–BMOP complex were calculated using Autodock program. Out of 10 conformers obtained, the conformer with the lowest binding free energy was used for further analysis. The crystal structure of BSA contain three homologous domains (I, II, and III): I (residues 1–183), II (184–376), III (377– 583), each containing two subdomains (A and B). The hydrophobic cavities in subdomain IIA and IIIA suggested as principle ligand binding site [53]. The best energy ranked model (Fig. 10A) revealed that the BMOP bound at the interface between two sub domains IIA and IIIA, which is located just above the entrance of the binding pocket of IIA. The amino acid residues lining this binding site are comprised of Arg196, Phe148, Arg458, Ser192, Gln195, His145, Tyr147 and Asp108 (Fig. 10B). HNBD and C120 also bind at this site of BSA [53]. The docking result indicates that the BMOP interact with BSA through hydrophobic interaction and single hydrogen bond between His 145 residue of BSA and O-atom of BMOP

Fig. 9. Infrared absorption, curve fitted and deconvoluted spectra of (a) free BSA (b) difference spectra of BSA–BMOP complex in the amide I band region (1600–1700 cm1) at 298 K and pH = 7.4.

M. Kumari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 349–356

355

Fig. 10. (A) Molecular docked model of BMOP (sphere representation) located within the hydrophobic pocket of BSA. (B) The surrounding amino acid residues of BSA within 5 A° from BMOP (red coloured). (C) Hydrogen bond represented using dashed lines (magenta) between O atom of BMOP (depicted in red stick) and His 145 residue of BSA (D) Distances (A°) between docked BMOP and Trp residues of BSA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Fig. 10C) in agreement with our conclusion of thermodynamic analysis. Conclusion The interaction between BSA and BMOP were studied using UV–Vis, FT-IR, fluorescence and time resolved spectroscopy and possible binding mechanism were confirmed through molecular docking method. The results obtained from time resolved and steady state spectroscopy shows that BMOP effectively quenched the intrinsic fluorescence of BSA through dynamic quenching mechanism. The binding constant and the number of binding site were calculated along with the thermodynamic parameters from van’t Hoff equation indicated that the hydrophobic interaction plays an important role in stabilizing the BSA–BMOP complex and the binding process is spontaneous, endothermic and entropy driven. UV–Vis spectra confirm the complex formation between the BMOP and BSA. The results obtained from Synchronous and FT-IR spectra indicated that the binding of BMOP to BSA induce the conformational change of BSA. In addition, molecular docking study confirms the interaction between the BMOP and BSA and consistent with available investigational data. This study on the interaction between BSA and BMOP help the understanding of the role that pyrrolidinium based ILs contributes to their application in the pharmaceutical industries in the future. Acknowledgment The authors thanks to Science and Engineering Research Board (SERB), New Delhi for providing research grant with Sanction Order No. (SR/S1/PC-19/2011). References [1] M.J. Earle, K.R. Seddon, Pure Appl. Chem. 72 (2000) 1391–1398. [2] C.M. Gordon, Catal. Appl. A Gen. 222 (2001) 101–117.

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