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Detailed Scrutiny of the Anion Receptor Pocket in Subdomain IIA of Serum Proteins toward Individual Response to Specific Ligands: HSAPocket Resembles Flexible Biological Slide-Wrench Unlike BSA Shubhashis Datta and Mintu Halder* Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India S Supporting Information *

ABSTRACT: Present study reveals that the subdomain IIA cavity of two homologous serum albumins (HSA, BSA) has inherent mutual structural and functional deviations which render noticeable difference in behavior toward specific ligands. The major drug binding site (subdomain IIA) of HSA is found to be largely hydrophobic while that of BSA is partially exposed to water. Larger shift in REE spectra and greater change in solvent reorganization energy of coumarin 343 (C343)-anion in HSA clearly reveals that binding pocket is relatively large and water molecules penetrate deeper into it unlike BSA. The individual response of proteins to perturbation by ligands is found to be way different. Although the subdomain IIA is primarily anion receptive (prefers anionic ligands), the present study suggests that HSA may also like to bind neutral guests due to its remarkable conformational features. Actually, HSA is capable of adopting favorable conformation like mechanical slide-wrench, when required, to accommodate neutral ligands [e.g., coumarin 314 (C314)], as well. But due to less flexible solution structure, BSA behaves like fixed mechanical spanners and hence is not very responsive to C314. Therefore, the generally speaking functional-structural similarities of homologous proteins can be apparent and needs to be analyzed exhaustively. affinity for the same ligand with two serum proteins.8,9 Although there are several reports on such differences in their behavior but no appropriate reason has been cited due to unavailability of crystal structure of BSA17 at the then moment. Recently the solution structures of HSA and BSA complexed with fatty acid (FA) have been investigated using double electron−electron resonance spectroscopy (DEER). It is reported that the functional solution structure of HSA has more symmetric distribution of entry point to the FA binding site than expected from the crystal structure, which indicates its increased surface flexibility and plasticity in solutions.18 On the other hand, the BSA conformational ensemble in solution seems to be closely related to the crystal structure. The authors have also concluded that the solution structure of HSA has more flexibility and conformational adaptability compared to that of BSA. The major functional role of serum proteins is transportation of endogenous and exogenous ligands via their initial temporary boarding into the pocket, which depends on the conformational ductility and adjustability of the host to facilitate the interaction with the guest. In another recent report, the effect of crowding agent on the local conformation around tryptophan residues in serum proteins has been investigated.19 It is reported that the individual response of

1. INTRODUCTION Water is an important medium in all biochemical processes.1 It has significant effects on the functional structure of proteins in solutions. Therefore, the investigation of the role of water in biomacromolecular studies2 is very important. Recently water molecules near extended hydrophobic protein surface have been found to significantly alter biomolecular interactions and functions.3 However, majority of these studies are performed from a global point-of-view. Few recent attempts on such physicochemical interactions between solvents and biomacromolecules have been made to look into the case sitespecifically.4−6 In protein research, serum albumins (SAs) with its high abundance in blood plasma,7 have been and still are model proteins for the study of protein−ligand8,9 and protein−solvent interactions.10,11 These are known to function as versatile transporter of various endogenous and exogenous ligands like drugs, dyes, and metals through the bloodstream.12−14 Human serum albumin (HSA) and bovine serum albumin (BSA) are the two mostly studied serum proteins.7 Although BSA has almost 88% sequence homology with HSA, their binding affinity for the same ligand is found to be invariably different.8,15 It is reported elsewhere that the binding affinity of serum albumins obtained from human, cat, dog, pig, and sheep is different for the same ligand, bromocresol green.16 In some of our recent studies on the interaction of food dyes with serum proteins, we have also observed difference in binding © 2014 American Chemical Society

Received: February 12, 2014 Revised: April 28, 2014 Published: May 15, 2014 6071

dx.doi.org/10.1021/jp501547r | J. Phys. Chem. B 2014, 118, 6071−6085

The Journal of Physical Chemistry B

Article

pocket-specific interaction of these proteins with any ligand is likely to be different in these two proteins. Here subdomain IIA binding ligands, coumarin 343 (C343)anion21 and its ethyl ester, coumarin 314 (C314, a neutral molecule)20 (shown in Scheme 1), have been purposely chosen

BSA and HSA to the crowder-induced perturbations is quite different and has been ascribed to be due to the discernible differences in their inherent plasticity. SAs are known to have a number of binding sites which can accommodate different molecules/ligands7 for transportation through bloodstream. There are plenty of reports available in the literature focused on the interaction of ligand at some specific site of SAs.8,9,12 In a recent report, we have pointed out that the subdomain IIA cavity of HSA acts as anion receptor.20 Also in another report, we have shown that prototropic equilibrium of coumarin 343 (C343) is considerably shifted toward the basic form due to preferential binding of the anionic form with HSA.21 The anion receptors available in the subdomain IIA of HSA preferably binds the deprotonated (anionic) form via electrostatic interaction and consequently binding of neutral/cationic ligands in this pocket is less favorable. A comparison of the amino acid sequence of both the serum proteins17 reveals that 15 amino acid residues in subdomain IIA of HSA have been replaced in the bovine variety (Table T1 of the Supporting Information). A closer examination of the active surface (Figure 1) clearly indicates that the said binding cavity of HSA is more open-type than in BSA. The strength of binding of a specific ligand in such a cavity is very much dependent on the nature of amino acid residues available there. It is interesting to note that BSA has more number of residues with positively charged side chains in that subdomain. Thus, the

Scheme 1. Structure of Coumarin 343-Anion (C343-anion) and Its Ethyl Ester, Coumarin 314 (C314)

to investigate the role of pocket-local solution structure and relative conformational flexibility/adaptability of these homologous proteins toward specific interaction. Coumarin dyes are frequently used as fluorescence probe for various photophysical studies due to their reasonably high fluorescence quantum yields, strong solvent polarity-dependent Stokes shift, and large change in dipole moment upon photoexcitation.22−24 These are used in different solvatochromic studies, determination of microenvironment of organized molecular-assemblies, photoinduced electron transfer, and solvent relaxation processes.22,23,25−28 When these subdomain IIA binder fluorophores enter the pocket from bulk aqueous phases their photophysics ought to be modulated due to the difference in the local details of two protein cavities. In the present study, we have used some specific approaches like study of red-edged effect (REE)29 and calculation of solvent reorganization energy.30 The major advantage of these approaches is that the information about the relative rates of solvent structural alterations and dynamics can be readily obtained, whereas other fluorescence techniques like fluorescence quenching, energy transfer, and polarization parameters provide information about the fluorophore itself. When exogenous ligand is accommodated in the protein pocket, the facility of the process can depend on the response of the macromolecule against the perturbation tendered by the guest. Naturally the reception offered by the host (protein) will differ due to the nature of the ligand and the capability of the former to house the latter. Herein we would like to address, how the receptability of the host could also be important in determining the strength of interaction particularly in reference to the subdomain IIA.

2. EXPERIMENTAL SECTION 2.1. Materials. HSA (>98%), BSA (∼99%), hemin (>98%, HPLC), and ibuprofen (>98%, GC) are purchased from SigmaAldrich and warfarin (analytical grade) is purchased from TCI chemicals, Japan. Coumarin 343 and coumarin 314 (laser grade, exciton) are also used as received. Sodium chloride (analytical grade); sucrose and tetra butyl ammonium bromide (TBAB); and 1,4-dioxane and ethanol (UV spectroscopic grade) from EMerck are used as received. Appropriate volume of stock NaCl solution (2 M) is added to protein-fluorophore solution in buffer to get various ionic strengths. Other chemicals are of analytical reagent grade and ultra pure water is used throughout the study for preparation of solutions. 2.2. Sample Preparation. Distilled ethanol is used for the preparation of fluorophore (C343 and C314) stock solutions. The required volume of fluorophore solution has been taken in

Figure 1. Active surface around the subdomain IIA cavity of (a) HSA and (b) BSA. Blue, red, and white portions indicate charge density arising due to positive, negative, and uncharged side chains of amino acid residues, respectively. 6072



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The data are fitted using Microcal ORIGIN. The values of heat change due to dilution of fluorophore in buffer are subtracted from the corresponding integrated ITC profile of proteinfluorophore titration.

a volumetric flask and then ethanol is evaporated followed by the addition of buffer solution to get the required concentration. The final concentration of fluorophore is kept at 1.5 μM throughout the experiment. All solutions are prepared in 5 mM sodium phosphate buffer of pH 7.4 (±0.1). EUTECH pH 510 ion pH meter is used for the measurement of pH. 2.3. Instrumentation and Methods. The UV−vis absorbance spectra have been recorded by scanning solutions on a Shimadzu UV-2450 absorption spectrophotometer against a solvent/buffer reference. The steady-state fluorescence spectra are recorded on a Jobin Yvon-Spex Fluorolog-3 spectrofluorimeter equipped with temperature-controlled water cooled cuvette holder. Quartz cuvette of 1 cm path length has been used. The steady-state fluorescence measurements have been performed at 300 K. During steady-state fluorescence measurements the excitation and emission slits are kept at 5 and 2 nm, respectively. Fluorescence quantum yield (Φf) has been determined by literature reported method31 using C343 as the secondary standard (Φf = 0.63 in ethanol32). Steady-state anisotropy measurements are carried out in a Horiba Jobin Yvon spectrofluorometer (Fluoromax 4). Fluorescence lifetimes are measured with the help of a single photon counting apparatus having an excitation source of 408 nm using a picosecond laser diode (IBH, Nanoled), and the signals are collected at the magic angle 54.7° using a Hamamatsu microchannel plate photomultiplier tube (R3809U), instrument response is 100 ps. Analysis of decays are done with IBH DAS, version 6 software. A χ2 value lying within range of 0.9−1.3 and weighted residuals ensures the perfectness of fit. During time-resolved anisotropy measurements, the samples have been excited at 408 nm using the same picosecond laser diode (IBH, Nanoled). A motorized polarizer has been used in the emission side. The emission intensities at parallel and perpendicular polarizations are collected alternately, until a certain peak difference has been reached between them. The peak differences depend on the tail matching of the parallel and perpendicular decays. The analysis of the data has also been done with above-mentioned software. Circular dichroism (CD) spectra have been recorded on a Jasco-810 automatic recording spectropolarimeter at 300 K over a wavelength range of 190−260 nm with a scan speed of 50 nm/min using a quartz cell having a path length of 0.1 cm. Each spectrum is an average of three successive scans. The CD spectra are corrected by subtracting the CD spectra of corresponding blank solution. The concentration of both HSA and BSA is kept at 9 μM, and the molar ratio of protein-to-fluorophore is varied as 1:0, 1:1, and 1:2. Molecular Docking Study. The binding location of C343anion in HSA and BSA has also been investigated using molecular docking simulation. Ligand has been docked into the crystal structure of HSA (PDB ID: 1AO6)33 and BSA (PDB ID: 4F5S).17 The detailed methodology of docking study9,20 is given in the Supporting Information. Isothermal Calorimetric Titration. The energetics of interaction of fluorophore with serum proteins has been determined using an isothermal calorimeter (iTC200, Microcal, Northampton, MA) at 300 K. The reference and reaction cell is filled with 5 mM phosphate buffer (pH 7.4) and solution of serum albumins (0.05 mM). A 0.56 mM fluorophore solution is added sequentially in 2 μL aliquots (for a total of 20 injections, 10 s duration each) at 120 s interval with stirring at 290 rpm.

3. RESULTS AND DISCUSSION 3.1. Modulation of Photophysical Properties in the Presence of Protein. With dependence on the pH of the medium, C343 (pKa = 4.6)21 can exists in one of the two prototropic forms, namely, neutral and anionic. Our experimental pH (pH 7.4) is much above the pKa and hence C343 remains predominantly as an anion. The fluorescence emission spectra of C343-anion at pH 7.4 with increasing concentrations of BSA is shown in Figure S1 of the Supporting Information and that in the presence of HSA is shown below. The emission maxima of C343-anion are blue shifted from 490 to 485 nm in the presence of BSA (Figure S1 of the Supporting Information) along with reduction of fluorescence quantum yield (from 0.57 to 0.46). On the other hand, in the presence of HSA, the emission maxima are blue-shifted from 490 to 479 nm (Figure 2) with substantial quenching of

Figure 2. Emission spectra of C343-anion in the absence and presence of increasing concentrations of HSA in pH 7.4 at 300 K. Top inset shows change in fluorescence intensity (ΔI) of C343-anion and C314 in the presence of serum proteins and bottom inset shows the Benesi− Hildebrand (B−H) plot for the complexation of C343-anion with serum proteins of the same pH and temperature.

luminescence (Φf decreases from 0.57 to 0.40). These observations indicate that both serum proteins bind C343anion and leads to the substantial changes in photophysical properties of the fluorophore. The more blue-shifted emission of C343-anion in HSA indicates that the binding pocket is deeply rooted in a less polar environment, whereas the same in BSA is somewhat exposed to the solvent. On the other hand, C314 does not show substantial changes both in fluorescence intensity and maxima in the presence of BSA, indicating weak interaction, whereas with HSA, the fluorescence intensity is considerably quenched with little alteration in emission maxima (Figure S2 of the Supporting Information). It is reported elsewhere that C343-anion binds the subdomain IIA cavity of HSA.21 The binding site in BSA has been confirmed via competitive binding studies by investigating the effect of known site-markers (warfarin, ibuprofen, and hemin) on the fluorescence property of protein-bound fluorophore, and it is found that only warfarin is able to displace the bound-C343-anion (Figure S3 of the Supporting 6073



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evident from very little changes in fluorescence data. Thus, it appears that there could be something special in the solution structure of HSA which makes it receptive to both anionic and neutral ligands. 3.2. Fluorophore-Serum Proteins Association Constant. C343-anion and C314 are known to form 1:1 complex with HSA.20,21 The binding stoichiometry for BSA:C343-anion complex is also found to be 1:1 by the modified Job’s method of continuous variation using absorbance spectroscopy9 (Shown in Figure S6 of the Supporting Information). The association constant of serum proteins with ligand is a measure of their transportability. The association process of the studied fluorophore with protein may be represented in the following Scheme 2, and the corresponding association constant (KB−H) is evaluated using the Benesi−Hildebrand (B−H) equation.15,34

Information). This means that C343-anion also binds the subdomain IIA of BSA. The change in fluorescence intensity (ΔI) of both the coumarin dyes as a function of protein concentration is shown in the inset (top) of Figure 2. ΔI initially rises followed by saturation after a certain concentration of protein. The saturation of fluorescence intensity is indicative of specific protein−ligand interaction. We have also measured the steady-state fluorescence anisotropy of both the fluorophore in the presence of serum proteins at the corresponding emission peaks. Fluorescence anisotropy gives a quantitative measure of the fluorophore-local fluidic property in various environments. An increase in the rigidity of the surrounding environment of fluorophore leads to the increase in fluorescence anisotropy.15,31 The variation of anisotropy as a function of protein concentration is shown below. From Figure 3, it is observed that with increase of protein the steady-state anisotropy initially rises followed by saturation.

Scheme 2. Complexation of Ligand with Serum Proteins in 1:1 Stoichiometry

Since the change in fluorescence intensity of C314 in the presence of BSA is considerably small, the determination of the association constant using the B−H equation has not been attempted there. The plot at pH 7.4 at 300 K using the B−H equation is shown in the inset (bottom) of Figure 2. The value of KB−H has been obtained from the slope of the above plot (shown in the bottom inset of Figure 2), and it is found to be on the order of 105, which indicates stronger complexation between fluorophore and serum proteins. Association constant (KB−H) for the HSA/C343-anion complex is (4.46 ± 0.02) × 105 M−1, which is higher than that for the BSA/C343-anion complex (1.12 ± 0.06) × 105 M−1 in pH 7.4 at 300 K. The association constant (KB−H) for the HSA/C314 complex is estimated to be (1.52 ± 0.01) × 105 M−1, under the same conditions. The magnitude of the association constant (KB−H) can be connected to the relative transportability of serum proteins. KB−H for the HSA/C343-anion complex is almost four times to that of the BSA/C343-anion complex, which indicates stronger affinity of HSA for the coumarin anion at pH 7.4. On the other hand, the association constant (KB−H) for HSA is considerably high with C343-anion than C314 and this is due to the fact that subdomain IIA of HSA is primarily an anion receptor (i.e., it prefers anionic ligands)20,21 and is likely to have lesser preference for neutral ligand like C314. Fluorescence anisotropy data can also be employed to determine the association constant (KAniso) between fluorophore and serum proteins.35 The magnitude of the association constant (KAniso) determined from the slope of the plot using anisotropy data (Figure S7 of the Supporting Information) are found to be (1.01 ± 0.1) × 105 M−1 and (3.15 ± 0.15) × 105 M−1 at 300 K for BSA: C343-anion and HSA:C343-anion complex, respectively, and the value of association constant (KAniso) of C314 with HSA is (1.15 ± 0.1) × 105 M−1. These

Figure 3. Variation of steady-state fluorescence anisotropy of C343anion and C314 with increasing concentrations of serum proteins in pH 7.4 at 300 K.

The anisotropy value of C343-anion at the point of saturation is found to be 0.23 and 0.15 with HSA and BSA, respectively. On the other hand, anisotropy at the point of saturation for C314 with HSA is found to be 0.11. The observed higher saturation anisotropy value of C343-anion with HSA indicates a more rigid environment in the binding nanocavity as compared to BSA. We have also investigated the effect of added salt on the aforementioned interactions. It is interesting to note that with increasing concentration of salt, the fluorescence intensity of the protein-bound C343-anion is enhanced along with red shifting of emission maxima (Figure S4 of the Supporting Information), which indicates the release of fluorophore from protein to the surrounding bulk medium. This effect is found at a lower salt concentration with BSA and at somewhat higher salt concentration with HSA indicating stronger electrostatic interaction in the latter case. On the other hand, the emission spectra of HSA-bound C314 remain unaffected by salt (Figure S5 of the Supporting Information). Salt can only influence the electrostatic complexation, but it remains inert to nonelectrostatic interactions. Thus, it looks like the binding mode of HSA with C314 is primarily nonelectrostatic in nature. Interestingly, the individual response of serum proteins to the incoming ligand structure is found to be quite different. HSA strongly binds both the fluorophores. On the other hand, BSA, which is homologous to HSA, is able to bind C343-anion strongly, but the interaction with neutral C314 is very weak as 6074



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also binding of the ligand at one site is dependent on its binding at the other site. The results of ITC measurements are tabulated in the following tables. Each parameter in the table is an average of three independent measurements. From Tables 1 and 2, it is observed that the value of the thermodynamic association constant (Ktherm) of HSA is higher with C343-anion. Binding of C343-anion with BSA is favored both enthalpically (negative enthalpy change) and entropically (positive entropy change). On the other hand, binding with HSA is favored enthalpically at both sites while the entropy change at one site is positive and at the other site is negative. The negative enthalpy changes correspond to either electrostatic or the hydrogen-bonding interaction.36 At pH 7.4, C343 remains as an anionic species and it should prefer to bind electrostatically with positively charged side chains of amino acid residues available in the pocket. Mobility/local motion of ligand complexed with protein is strongly retarded when it is bound by hydrogen bonding than by electrostatic interaction. This is because electrostatic interaction is basically a charge− charge interaction occurring through space and does not lead to real bond formation, and there remains an option for the ligand to wiggle.37 Apparent discrepancy in the value of binding site number determined from fluorimetric and calorimetric methods has also been previously reported elsewhere.38 As the calorimetric titrations are performed at high concentration compared to that used in fluorimetric measurements, the existence of nonspecific interactions with amino acid side chains distant from the tryptophan residue in the binding pocket has been attributed to the second binding site. In those cases both fluorimetric and isothermal titration of serum proteins are carried out by increasing concentration of ligand.38 But in our case, although we have performed isothermal calorimetric titration of serum proteins by ligands, the steady-state fluorescence titrations are performed in the reversed way by increasing the concentration of serum proteins to a fixed concentration of coumarins (ligand). The steady-state fluorescence titrations have been purposely arranged to probe the microenvironment around the bound fluorophore. Therefore, if a ligand has two different sites, where binding at one site is stronger than the other, then in the presence of excess concentration of proteins it will bind the high-affinity site, and this is probably why we are observing a single binding site in steady-state fluorescence study. This is merely a means to identify the most affinity site of C343-anion

values of association constants (KAniso) are nearly equal to those (KB−H) obtained from the fluorescence data using B−H plot. 3.3. Thermodynamics of Protein Ligand Association. The association of ligand with serum protein can be via various intermolecular weak forces such as electrostatic, hydrophobic, hydrogen-bonding, and van der Waals interactions.8,9 Ross and Subramanian have reported that signs and magnitudes of the thermodynamic parameters (ΔH0 and ΔS0) can account for the predominant forces involved in the binding process.36 The thermodynamic parameters for binding of C343-anion with both serum proteins have been determined from ITC measurements. Calorimetric profiles at pH 7.4 for the titration of serum proteins by C343-anion at 300 K are shown in the following figures. Each peak in the upper panel of Figure 4 (panels a and b) represents a single injection of ligand into the protein solution.

Figure 4. ITC profile for the titration of 0.05 mM (a) HSA and (b) BSA by 0.56 mM C343-anion in pH 7.4 at 300 K. Each peak in the upper panel indicates each single injection and lower panel shows an integrated heat profile against a molar ratio. The solid line represents the best nonlinear least-squares fit.

The lower panel of the plot (Figure 4, panels a and b) represents heat change as a function of mole ratio of the C343anion/serum protein. The data are best-fitted by using a onesite binding model for the titration of BSA by C343-anion and using a two-site sequential binding model for the titration of HSA by the C343-anion. The two-site sequential binding model indicates the presence of two different binding sites and

Table 1. Thermodynamic Parameters for the Binding Interaction of BSA with C343−Anion in Absence and Presence of Different Additives in pH 7.4 at 300 Ka

a

additives

Ktherm

ΔH0 (kJ mol−1)

ΔS0 (J mol−1 K−1)

(i) no additive (ii) NaCl (a) 0.02 M (b) 0.05 M (c) 0.1 M (iii) Sucrose (a) 0.2 M (b) 0.4 M (c) 1 M (iv) TBAB (a) 0.5 M (b) 1.0 M

(6.96 ± 0.22) × 104

(−2.93 ± 0.25)

(83.16 ± 0.41)

(3.35 ± 0.21) × 104 (2.14 ± 0.52) × 104 n.o.

(−8.58 ± 0.14) (−14.34 ± 0.29) n.o.

(58.14 ± 0.13) (35.21 ± 0.26) n.o.

(6.58 ± 0.03) × 104 (6.25 ± 0.06) × 104 (5.98 ± 0.21) × 104

(−5.54 ± 0.09) (−9.85 ± 0.14) (−11.89 ± 0.26)

(73.77 ± 0.25) (58.96 ± 0.65) (51.8 ± 0.37)

(6.14 ± 0.34) × 104 (5.45 ± 0.26) × 104

(−7.55 ± 0.18) (−12.34 ± 0.96)

(66.46 ± 0.65) (49.48 ± 0.56)

n.o. = any reasonable ITC profile is not observed. 6075



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Table 2. Thermodynamic Parameters for the Binding Interaction of HSA with C343−Anion in Absence and Presence of Different Additives in pH 7.4 at 300 Ka

a

ΔH10 (kJ mol−1)

ΔH20 (kJ mol−1)

ΔS10 (J mol−1 K−1)

ΔS20 (J mol−1 K−1)

4

(9.16 ± 0.73) × 10

(−10.15 ± 0.60)

(−30.53 ± 0.6)

(63.84 ± 0.65)

(−7.01 ± 0.46)

(9.8 ± 0.03) × 104 (6.85 ± 0.75) × 104 (4.65 ± 0.13) × 104

(9.84 ± 0.72) × 104 (1.16 ± 0.33) × 105 (1.45 ± 0.57) × 105

(−15.84 ± 0.63) (−21.58 ± 0.34) (−25.55 ± 0.18)

(−32.5 ± 0.32) (−36.1 ± 0.31) (−39.5 ± 0.89)

(42.68 ± 0.14) (20.63 ± 0.21) (4.18 ± 0.54)

(−12.7 ± 0.48) (−23.4 ± 0.34) (−32.8 ± 0.77)

(1.28 ± 0.08) × 105 (1.34 ± 0.11) × 105 (1.41 ± 0.14) × 105

(7.95 ± 0.32) × 104 (6.84 ± 0.21) × 104 (5.12 ± 0.55) × 104

(−9.85 ± 0.77) (−8.5 ± 0.98) (−8.15 ± 0.26)

(−26.32 ± 0.84) (−21.65 ± 0.98) (−18.78 ± 0.11)

(64.93 ± 0.76) (69.8 ± 0.65) (71.4 ± 0.89)

(6.07 ± 0.58) (20.39 ± 0.98) (27.55 ± 0.66)

(1.08 ± 0.11) × 105 (9.38 ± 0.25) × 104

(9.65 ± 0.12) × 104 (9.94 ± 0.21) × 104

(−12.8 ± 0.84) (−17.6 ± 0.45)

(−32.17 ± 0.59) (−34.8 ± 0.35)

(53.66 ± 0.97) (36.4 ± 0.63)

(−11.7 ± 0.19) (−20.3 ± 0.76)

additives

(Ktherm)1

(i) no additive (ii) NaCl (a) 0.02 M (b) 0.05 M (c) 0.1 M (iii) Sucrose (a) 0.2 M (b) 0.4 M (c) 1 M (iv) TBAB (a) 0.5 M (b) 1.0 M

(1.25 ± 0.13) × 10

(Ktherm)2 5

Subscripts 1 and 2 represent the corresponding value for sites 1 and 2, respectively.

In the steady-state fluorescence experiment, we have also studied the effect of addition of salt, sucrose, and TBAB on HSA/BSA:C343-anion complex. It is observed that only salt is able to displace the bound C343-anion from protein. We have also explored the effect of salt on the fluorescence property of C343-anion, and it is found that neither its emission maxima nor the fluorescence quantum yield is affected in our experimental salt concentrations. Therefore, the results clearly indicate that the major force of binding with both proteins is electrostatic in nature. Hence, out of the two binding sites of C343-anion, as appeared in the ITC measurements, the site having electrostatic contribution with comparatively a higher binding constant is the major affinity site. Enthalpy−entropy compensation (EEC) is an important phenomenon observed in many chemical and biochemical processes involving weak forces where the change in enthalpy is compensated by the corresponding change in entropy in such a way that the net free-energy change of the system remains almost unaltered;41−43 that is, there is hardly any change in the binding constant. This phenomenon is also observed in drugreceptor binding where either change in any external factors like temperature or any perturbation to the existing mode of interaction is reflected in the entropy and enthalpy changes in opposite direction to each other.44 Tighter binding of drug within the receptor cavity results in decrease of mobility or local motion (consequent decrease in entropy) of the former which is compensated by the increase in enthalpy.45 Thus, the mechanism of enthalpy−entropy compensation is very much system-dependent. As serum proteins function as transporter of various crucial pharmaceuticals, exploration of the molecular basis of their binding with ligands from thermodynamics is important in biophysical research. With dependence on the modes of interaction, the affinity of serum proteins toward any ligand can be modulated by means of external additives containing hydrogen-bonding groups (e.g., sucrose), hydrophobic groups (e.g., TBAB), and salt (e.g., NaCl). With these external perturbations, the thermodynamics of ligand binding can be modified. In a recent study, we have observed nice enthalpy−entropy compensation for complexation of a fooddye, tartrazine, with BSA and HSA in the presence of various ionic strength of the medium.37 That study nicely demonstrated that by varying the amount of added salt, one can control the release of ligand from serum proteins which can be useful for purification of proteins mediated by such ligands.

in HSA out of two binding sites as obtained from ITC measurements. Binding in the Presence of Salt. Electrostatic interaction between ion pairs is affected by the alteration of the ionic strength of the medium. Thus, contribution of electrostatic interaction in protein−ligand association processes can be understood from the study of binding thermodynamics in solutions in the presence of different added salt concentrations. Several research groups have studied the effect of salt on electrostatic complexation in micellar environment, vesicles, and even in hydrophobic protein environments.39,40 In those cases, salt can even penetrate into the hydrophobic environment through water channels and rupture the electrostatic association there inside. To investigate role of electrostatic interaction in the binding of C343-anion with the serum proteins, we have performed calorimetric measurements in the presence of three different salt concentrations, 0.02, 0.05, and 0.1 M NaCl, and the parameters obtained from these measurements are listed in Table 1 and Table 2. From Table 1, it is observed that thermodynamic association constant (Ktherm) of BSA with C343-anion is decreased with increasing salt concentration, and at 0.1 M salt concentration, binding becomes so weak that no reasonable titration profile is generated. These results indicate that electrostatic interaction plays a major role in the binding of BSA with C343-anion, which is disrupted in the presence of NaCl. On the other hand, Ktherm for the HSA:C343-anion complex (from Table 2) at one site is decreased whereas at the other site is very slightly increased with salt concentration. These observations indicate that binding of C343-anion with HSA at one site is controlled by electrostatic interaction and hence becomes weak with increasing salt concentration. The binding at the other site is nonelectrostatic and weak in nature, but with increasing ionic strength of the medium it becomes a major binding site as electrostatic contribution at the other site fades out. The decrease of electrostatic interaction with increasing ionic strength of the medium is also well-understood from sharp reduction in the magnitude of entropy changes. Here the entropy change becomes less positive at high salt concentration. As the increase of ionic strength insulates charge−charge interaction between ligand and protein, the other nonelectrostatic modes like hydrogen-bonding and van der Waals forces become major players, which results in the less positive entropy changes.37 6076



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enthalpy and entropy. From the effect of salt variation, it is clear that the most affinity sites in HSA (having higher binding constant) binds the C343-anion by electrostatic interaction primarily. Therefore, binding of C343-anion at the other site is controlled by hydrogen-bonding interactions. Binding in the Presence of Tetrabutyl Ammonium Bromide (TBAB). Tetrabutyl ammonium bromide contains four bulky butyl groups, which can alter the strength of hydrophobic interaction between protein and the ligand. As it is a salt, it may also insulate charge−charge interaction. But its primary role should be to influence the hydrophobic interaction.47 Therefore, in order to investigate role of hydrophobic interaction in binding of coumarins, calorimetric titrations of serum proteins by C343-anion are performed in the presence of two different concentrations of TBAB, and the results obtained from these experiments are shown in Tables 1 and 2. From Table 1, it is observed that the thermodynamic association constant (Ktherm) of BSA with C343-anion is slightly reduced with increasing concentration of TBAB. Also the change in both binding enthalpy and entropy is very small. On the other hand, Ktherm of C343-anion at its major affinity site in HSA (observed from Table 2) is slightly reduced, while the value corresponding to the other site remains unaltered. Therefore, a small decrease in the value of Ktherm in the presence of TBAB indicates that the contribution of hydrophobic interaction is negligible for both proteins. As TBAB is salt, it is also possible that the slight decrease in the magnitude of the Ktherm with increasing concentration could be due to reduction of electrostatic interaction between protein and ligand. Thermodynamics of Binding Interaction of HSA with C314. All calorimetric titrations are performed at high concentrations of ligand (0.56 mM). Such high concentration of C314 (a neutral molecule) in phosphate buffer cannot be achieved and hence ITC measurements are not attempted there. Rather its thermodynamics of binding has been determined fluorimetrically titrating with HSA at three different temperatures, namely, 288, 298, and 308 K. The association constants (KB−H) obtained using the B−H equation15 from those fluorimetric titrations are plotted by the van’t Hoff method.8,9 The thermodynamic parameters are determined from the slope and intercept of the linear fitted plots and are shown in Table T2 of the Supporting Information. From Table T2 of the Supporting Information, it is observed that binding of C314 with HSA is associated with negative enthalpy change and negative entropy change, which means that binding is enthalpically favorable but entropically hard. As mentioned earlier, the negative enthalpy change may correspond to either electrostatic or hydrogen-bonding interaction, but the negative entropy change rules out the possibility of electrostatic interaction. C314 is a neutral coumarin. It has a carbonyl group which can take part in hydrogen bonding with nearby amino acid side chains,48,49 and also its nonpolar hydrocarbon part may undergo hydrophobic interaction within the pocket.48,49 Therefore, the possible mode of binding interaction of C314 with HSA can be hydrogen bonding and/or a hydrophobic interaction. We have also studied the effect of addition of salt, sucrose, and TBAB on the HSA:C314 complex. It is observed that sucrose and TBAB are able to release the coumarin fluorophore from the HSA−C314 complex, and also it has been mentioned in the earlier section that the fluorescence spectra of HSA-bound C314 remain

In our present study, as observed from the results of the ITC measurements, the complexation of C343-anion with BSA is found to be driven by electrostatic force. On the other hand, the complexation with HSA is biphasic, at one of the binding sites, the interaction is primarily guided by electrostatic force, whereas in the second site, it is by and large guided by nonelectrostatic forces. The plot of enthalpy change (ΔHo) against entropy change at a constant temperature (TΔSo) for binding of C343-anion with BSA and that for the interaction of C343-anion at the major-affinity site with HSA at different ionic strengths is shown in Figures S8 and S9 of the Supporting Information, respectively. Linear correlations of both plots indicate the enthalpy−entropy compensation. The slopes of both plots are very close to unity (0.89 ± 0.02) and (0.90 ± 0.04) for BSA and HSA, respectively. A closer look at the thermodynamic parameters in both cases (Tables 1 and 2) reveals that in the absence of salt, the entropic contribution is positive. With increasing salt concentrations, the entropic contribution becomes less positive and also the exothermic enthalpy term increases. Thus, in the absence of salt, the interaction is dominated by the electrostatic interaction and with increasing salt concentration, the nonelectrostatic mode of interactions start to manifest. The contribution of electrostatic interaction for the binding of C343-anion at the major affinity site in HSA persists, even at a 0.02 M salt concentration, while the contribution of electrostatic mode for the BSA:C343-anion complex is greatly decreased at this salt concentration. This result indicates stronger ion-pair formation in the case of the HSA:C343-anion system. In the presence of high salt concentrations, the nonelectrostatic mode of interactions begins to show up and, hence, the ligand binds more tightly resulting in the loss of entropy. The loss of entropy is compensated by the concomitant gain in enthalpy. Binding in the Presence of Sucrose. The role of hydrogenbonding in protein−ligand association can be investigated by performing calorimetric titration of protein with ligand in the presence of sucrose. Sucrose can form hydrogen bonds and therefore affects the hydrogen-bonding interaction between the protein and the ligand. We have performed calorimetric titration in the presence of three different concentrations of sucrose, namely, 0.2, 0.4, and 1 M, and the results obtained from these measurements are given in Tables 1 and 2. With increasing concentration of sucrose, the thermodynamic association constant (Ktherm) corresponding to binding of BSA with C343-anion (observed from Table 1) is found to be marginally affected, but the binding turns to be more exothermic with reduction in the magnitude of entropy change. The constancy of Ktherm with increasing concentration of sucrose indicates that hydrogen bonding does not play a major role in the binding of the C343-anion with BSA.46 On the other hand, a slight increase of the exothermicity of binding indicates the alteration in the solvent structure. Sucrose can form hydrogen bonds with the amino acid side chains of protein. Thus, increasing sucrose concentration results in formation of more number of hydrogen bonds leading to negative enthalpy change.46 This is also complemented by a decrease in entropy. The thermodynamic association constant (Ktherm) of C343anion at one of the binding sites in HSA (observed from Table 2) is decreased with increasing concentration of sucrose. The enthalpy change at this site becomes less exothermic with little increase of entropy. On the other hand, Ktherm at the second site remains almost the same with little alteration in both binding 6077



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unaffected by salt, which again shows that the major forces of binding with HSA are hydrogen bonding and hydrophobic interaction. 3.4. Red-Edged Effects (REE). Shift of emission maxima with change of excitation wavelength and failure of excited-state energy-transfer at the red-end of the absorption spectrum in the case of organic aromatic fluorophores in different rigid and viscous media has been first discovered in the year 1970.50 This phenomenon which violates Kasha’s rule for electronic transition is commonly known to be “red-edged effects” (REE).29 The idea has been developed from the conclusive role in all these phenomena of inhomogeneous broadening of spectra, which mainly arises due to distribution of solvent− solute interaction energy.29 Fluorophores in the protein pocket are in contact with different environments, which results in difference in interaction energies. This leads to the formation of the ensemble of substates. These substates have different sharp spectral maxima, and when the average is taken, the inhomogeneous broadening of the spectra is observed. This type of broadening is found to be characteristic for fluorophores having greater dipole moment in the excited state than in the ground state.29 The other important criteria for showing REE is that the solvent relaxation (reorientation) time must be comparable to or longer than the lifetime of the excited fluorophore so that emission from different unrelaxed fluorophores gives rise to the excitation wavelength dependent fluorescence.51 The nonbonding interactions like hydrogen bonding and electrostatic force are also found to be responsible for such broadening of spectra in few cases.52 Here, solvent relaxation dynamics includes dynamics of restricted solvent (water) and also dynamics of the host dipolar matrix such as the peptide backbone of proteins. We have monitored the emission maxima of fluorophore with shifting of excitation wavelength toward the red end of the absorbance spectra at different concentrations of protein. The plots of REE of the coumarin dyes in the presence of proteins are shown below. Due to the shift of excitation quantum to the longer wavelength region, the emission maxima of C343-anion are also shifted to the red end (Figures 5 and 6a) and a limiting value of

Figure 6. Fluorescence emission spectra of (a) C343-anion and (b) C314 as a function of excitation wavelength in the presence of HSA in pH 7.4 at 300 K.

Binding in one site is guided by electrostatic interaction, while in the other site by hydrogen bonding interaction and corresponding interaction energies are also different. Hence, the distribution of solvent-fluorophore interaction energy should be inhomogeneous to a larger extent as is reflected in the higher REE. On the other hand, C343-anion has only one binding site in BSA, which is guided by electrostatic interaction. Because of the single binding site, the distribution of solventfluorophore interaction energy should be inhomogeneous to a lesser extent, which is also manifested by lower REE. Both C343-anion and C314 bind in subdomain IIA of HSA. Here the interesting observation is that unlike the case of C343anion, emission maximum of HSA-bound C314 is almost independent of the excitation wavelength (Figure 6b). Although C314 possess a considerably higher ground-state dipole moment (∼8D)53 and upon photoexcitation its dipole moment increases by 4 units,54 it does not show REE in the presence of HSA. The subdomain IIA of both serum proteins predominantly acts as an anion receptor, and hence, it prefers to bind anionic ligands by electrostatic interaction. In some cases, higher REE arises from the inhomogeneous distribution of solvent−solute interaction energy and has been attributed to the involvement of electrostatic interaction; on the other hand, involvement of nonelectrostatic modes results in lower REE.55 The thermodynamic parameters reveal that C314 binds the subdomain IIA of HSA by hydrogen bonding and hydrophobic interaction. Thus the nonelectrostatic modes of binding should result in a more uniform distribution of solvent−fluorophore interaction energy, which is evident from very little REE in the case of HSA. Exploration of structure and energetics of solvent molecules in the hydrophobic cavity/cleft of proteins have been an involved problem in biomolecular research.56−58 Several research groups have reported that the dynamic behavior of water molecules in the hydrophobic cavities of protein is not unique but very much case-specific and depends on the structural and functional parameters of proteins.59 Binding site of an anticancer drug doxorubicin in HSA is ∼10−12 Å deep seated from the protein surface where water molecules are also available.60 Recently it has been reported that the water dynamics in the hydrophobic cavity of subdomain IIA of HSA is considerably slowed down even up to ∼600 ps.61 REE helps to understand the rotational mobility of the environment itself where the employed fluorophore acts as reporter agency.62 REE depends on the environment-induced motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore.62 Thus, higher REE of HSA:C343-anion complex reveals that C343-anion is deeply rooted in the cavity

Figure 5. Variation of emission maxima of C343-anion as a function of excitation wavelength in the presence of different concentrations of BSA in pH 7.4 at 300 K.

the emission peak position is observed at 492 nm. Figure 6a shows how the emission maximum of HSA-bound C343-anion is shifted to the longer wavelength. C343-anion shows higher REE in the presence of HSA than with BSA, which indicates larger inhomogeneous distribution of C343-anion in the former. It is evident from the thermodynamic measurements that C343-anion has two different binding sites in HSA. 6078



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300 K are estimated using the standard equation.31 The average fluorescence lifetime (τavg) has been calculated using the procedure as described elsewhere9,12and are tabulated in the following Table 3. From Table 3, it is also clear that average lifetime of C343anion is reduced in the presence of HSA, whereas with BSA, the lifetime is very less affected. Thus, it indicates that the microenvironment around the binding site of C343-anion in HSA is considerably different from the bulk water (i.e., the pocket is deep-rooted). On the other hand, the microenvironment around C343-anion in BSA is only slightly different compared to the bulk solvent (i.e., binding site should be exposed to water). On the other hand, the average fluorescence lifetime of C314 is not greatly decreased in the presence of HSA, which also indicates that the microenvironment experienced in the pocket does not differ drastically from that in the bulk solvent. In earlier sections, we have discussed that the solution structure of HSA has some special feature which makes it more receptive to both anionic and neutral ligands. It is reported in the literature that conformational flexibility in the solution structure is special to HSA,18 and our present observations are also in-line with the same fact that possibly due to conformational adjustment, HSA may imbibe plausive conformation to bind neutral ligands like C314, although it prefers to have anionic ligands in general. During such conformational adaptation, the altered binding pocket in HSA becomes more accessible to water and, hence, the lifetime of the fluorophore is not altered considerably. The change in radiative and nonradiative decay constants (Table 3) as a function of protein concentrations is consistent with steadystate fluorescence quenching data. From Table 3, it is observed that the radiative decay constant for the slower component decreases with increasing protein concentration, whereas the same for the faster component remains almost unchanged, although the nonradiative decay constant for both the components increases. The magnitude of bimolecular quenching constant (kq) for complex formation has been obtained from the plot of τ0/τ vs protein concentration (Figure S11 of the Supporting Information) using the standard equation.64 Here τ0 and τ are the average lifetimes of fluorophore in the absence and presence of protein, respectively. The calculated value of kq is on the order of 1012 M−1 s−1. The values are quite large than the limit imposed by the diffusional motion under the same condition (2 × 1010 M−1 s−1).64 Therefore, the mechanism of quenching is static, that is, via ground-state complexation. Time-Resolved Fluorescence Anisotropy. Time-resolved fluorescence anisotropy is also another important technique

where the mobility of solvent is greatly restricted, and hence, a more rigid environment is experienced. 3.5. Time-Resolved Measurements. Time-Resolved Fluorescence Decay. Time-resolved fluorescence study is an important and very sensitive technique to monitor the change in the microenvironment of fluorophores.31 It is also helpful to look into various excited state phenomena.31 The change in fluorescence lifetime of fluorophore in the protein cavity can also provide useful ideas about the local environment. From the steady-state fluorescence measurements, it is clear that the binding strength of BSA with C314 is very weak, hence, we have performed the fluorescence lifetime measurements of C314 in the presence of HSA only (see Figure S10 of the Supporting Information). The decay profile of C343-anion at its emission maxima against various concentrations of both HSA and BSA at pH 7.4 are shown below. From Figure 7a, it is observed that the fluorescence decay of C343-anion is affected in the presence of HSA, whereas with

Figure 7. Fluorescence decay profiles of C343-anion in the absence and presence of (a) HSA and (b) BSA in pH 7.4 at 300 K. λex = 408 nm and λem = emission maxima. [C343-anion] = 1.5 μM.

BSA, change is comparatively less (Figure 7b). The lifetimes are obtained by fitting the decay profile and are tabulated in Table 3. Both C343-anion and C314 show single exponential decay in bulk water.63 In the presence of protein, the decay plot becomes biexponential with short and long components. The multicomponent decay of fluorophore in the presence of protein is a common observation.31 As the addition of serum protein decreases fluorescence lifetimes of both the coumarin dyes, it is quite expected that the shorter (faster) component will be due to the fluorophore bound to SA, and the longer (slower) component could be due to the free or unbound form of the fluorophore.31 The rate constant for radiative (kr) and nonradiative (knr) decay processes for both shorter and longer component at various concentrations of proteins in pH 7.4 at

Table 3. Time-Resolved Decay Parameters and Radiative and Nonradiative Decay Constants of C343−Anion and C314 in the Absence and Presence of Serum Proteins in pH 7.4 at 300 K

C343-anion only in buffer C343-anion + 10 μM HSA C343-anion + 15 μM HSA C343-anion + 30 μM HSA C343-anion + 15 μM BSA C343-anion + 30 μM BSA C314 only in buffer C314 + 15 μM HSA C314 + 30 μM HSA

τ1 (ns) (α1)

τ2 (ns) (α2)

4.47 4.27 4.21 4.14 4.42 4.39 4.03 3.97 3.93

− 1.75 1.56 1.46 1.25 0.82 − 0.83 0.87

(1.00) (0.71) (0.69) (0.68) (0.90) (0.88) (1.00) (0.8) (0.75)

(0.29) (0.31) (0.32) (0.10) (0.12) (0.2) (0.25)

τavg (ns)

χ2

quantum yield (Φ)

4.47 3.53 3.42 3.27 4.11 3.95 4.03 3.38 3.20

1.10 1.11 1.12 1.11 1.03 1.05 0.93 0.94 1.01

0.57 0.44 0.40 0.38 0.51 0.46 0.57 0.44 0.40 6079

kr (s−1) (slow) 1.27 1.03 9.50 9.17 1.15 1.04 1.27 1.03 9.50

× × × × × × × × ×

108 108 107 107 108 108 108 108 107

kr (s−1) (fast) − 2.51 2.56 2.60 4.08 5.60 − 2.51 2.56

× × × × ×

knr (s−1) (slow) 8

10 108 108 108 108

× 108 × 108

9.61 1.31 1.42 1.49 1.10 1.23 9.61 1.31 1.42

× × × × × × × × ×

107 108 108 108 108 108 107 108 108

knr (s−1) (fast) − 5.71 6.41 6.84 8.00 1.22 − 5.71 6.41

× × × × ×

108 108 108 108 109

× 108 × 108



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3.6. Micropolarity and Microviscosity of Binding Pocket. Micropolarity is generally expressed in ET (30) scale.15,67 The emission spectra of C343-anion and C314 in different mixed-solvent having varying percentage of water and dioxane are measured. Comparing the emission maxima of C343-anion and C314 in the protein environment with those in the mixed solvent of known ET (30)68 values, the polarity around the microenvironment of binding pocket of serum protein can be ascertained. It is observed that the micropolarity in the binding pocket of C343-anion in HSA and BSA is around 46.7 and 49, respectively, which indicates that C343-anion is located in a less polar environment in HSA, and in BSA it is more exposed to water (i.e., in more polar environment). On the other hand, the micropolarity around C314 in HSA is found to be close to 48.3. Microviscosity is one of the important internal properties of micelles and extended studies have been carried out to measure this property in the last few decades.69,70 With the help of timeresolved fluorescence anisotropy data, the microviscosity of the binding pocket of coumarin dyes have been calculated using Debye−Stokes−Einstein equation.71 The estimated values of microviscosity around the binding site of C343-anion and C314 in serum proteins are shown in Table 4. From Table 4, it is found that the microviscosity experienced by C343-anion and neutral C314 in aqueous buffer of pH 7.4 are 2.12 and 1.25 cP, respectively, which are closer to the value of microviscosity of water (1 cP) at room temperature. With the addition of protein, the microviscosity experienced by the fluorophore dramatically increases, indicating the entrance of coumarins from the bulk aqueous phase to the viscous protein pocket. The microviscosity for C343-anion around its binding site in HSA is higher than in the corresponding BSA pocket. On the other hand, microviscosity experienced by neutral C314 in HSA is lower. The shift of emission maxima, REE, both steady-state and time-resolved anisotropy measurements and micropolarity estimation studies have shown that the location of C343-anion is in the hydrophobic core of subdomain IIA of HSA, whereas the location in BSA is in a less hydrophobic and little water exposed outer part (discussed in the earlier section) of subdomain IIA. Thus, microviscosity experienced by both the coumarin dyes near their respective binding pocket is supportive to this. 3.7. Circular Dichroism (CD) Spectroscopy. CD spectroscopy is a sensitive technique to monitor the secondary structural changes of protein upon interaction with the ligand.8,9,37 CD spectra of BSA and HSA in the absence and presence of C343-anion at pH 7.4 are shown in Figure 9 (panels a and b). The CD spectra of HSA in the presence of

to gather information about the microenvironment of the fluorophore in different microheterogeneous systems like protein, molecular aggregates.48,65 We have measured the time-resolved fluorescence anisotropy decay in the absence and presence of different concentration of proteins at pH 7.4 and are shown below. Both C343-anion and C314 exhibit single exponential anisotropy decay in aqueous buffer (Figure 8, panels a and b,

Figure 8. Time-resolved fluorescence anisotropy decay of C343-anion in the absence and presence of various concentrations of (a) HSA and (b) BSA in pH 7.4 at 300 K. [C343-anion] = 1.5 μM.

and Figure S12 of the Supporting Information). In the presence of protein, the anisotropy decay profile becomes biexponential with two distinct correlation times (one fast and another slow component). The biexponential anisotropy decay in proteins may be expressed in terms of r0, α, and τ by the literature reported method.66 r0 is the limiting anisotropy that corresponds to the inherent depolarization of the fluorophore. α and τ are the pre-exponential factors and rotational correlation time, respectively, obtained from the fitting of anisotropy decay profiles.66 The average rotational correlation time has been calculated applying the known equation reported elsewhere.31 The parameters obtained from time-resolved anisotropy decay are tabulated as follows. From Table 4, it is observed that the value of average rotational correlation time increases with addition of protein. A substantial increase in rotational correlation time of the C343anion is observed in the presence of HSA as compared to BSA. These results are similar to those observed in the steady-state anisotropy measurements and indicate that C343-anion experiences a more rotationally restricted environment in the binding pocket of HSA. On the other hand, change in rotational correlation time of C314 in HSA is smaller, and it indicates that in order to accommodate C314, the ligand perturbed binding cavity becomes more within the reach of water.

Table 4. Anisotropy Decay Parameters in Absence and Presence of Serum Proteins in pH 7.4 at 300 K C343-anion only in buffer C343-anion + 7.5 μM HSA C343-anion + 22.5 μM HSA C343-anion + 7.5 μM BSA C343-anion + 22.5 μM BSA C314 only in buffer C314 + 15 μM HSA C314 + 30 μM HSA

αfast

τfast (ns)

αslow

τslow (ns)

(ns)

microviscosity(η)(cP)#

1 0.75 0.37 0.89 0.76 1 0.71 0.64

0.22 0.20 0.15 0.23 0.23 0.15 0.16 0.17

− 0.25 0.63 0.11 0.24 − 0.29 0.36

− 8.84 7.68 8.32 8.83 − 3.43 3.45

0.22 2.35 4.90 1.09 2.24 0.15 1.12 1.37

2.1 40.3 84.1 18.7 38.4 1.3 19.2 24.5

#

Error in estimation: ±5%. 6080



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3.8. Calculation of Solvent Reorganization Energy. Reorganization energy (RE) is an important parameter used to study solvation dynamics and solvent relaxation. It is a measure of the strength of the interaction of chromophores with surrounding solvent media.30 RE is generally expressed as half of the Stokes shift.30,75 The chromophore which shows polarity-dependent Stokes shift can be employed to calculate RE.76 In such chromophores, the variation of local motion strongly influences its electronic energy levels, leading to the change in magnitude of the Stokes shift. For protein-bound chromophore, the motion of amino acid residues/side chains and water molecules around its binding site can also have strong influence on the magnitude of Stokes shift. C343 and C314 are well-known polarity sensitive fluorophore, and there exists a reasonable difference between their ground-state and excited-state dipole moments (∼5−8 units).76 Polarity-dependent Stokes shift of Na-salt of C343 has been utilized to determine CMC of the CTAB micelle.76 We have calculated the reorganization energy at pH 7.4 by applying a literaturereported procedure.75 The change in the values of RE of the HSA/BSA:C343-anion complex is shown in the following Figure 10 and that of the HSA:C314 complex is shown in Figure 11.

C314 are shown in the Figure S13 of the Supporting Information.

Figure 9. CD spectra of (a) HSA and (b) BSA with increasing concentration of C343-anion in pH 7.4 at 300 K. [HSA] = [BSA] = 9 μM.

α-helix contents of free and complexed HSA or BSA are calculated by the literature-reported methods.9 The values of αhelix contents for free HSA and BSA at 300 K are (67.94 ± 0.95)% and (66.08 ± 0.63)%, respectively, at pH 7.4. In the presence of C343-anion α-helix content of HSA is increased by 2.1 units (∼3.1% increase with respect to protein without ligand) and that of BSA is by 1.5 units (∼2.5% increase). On the other hand, increase in α-helix content of HSA in the presence of C314 is 2.7 units (∼4% increase). In order to respond to the perturbation by the ligand, HSA adopts more flexibly optimal conformation to bind neutral C314, while BSA being less elastic fails to do so. Thus, due to ligand perturbation, the secondary structure of HSA is found to be altered little more compared to BSA. Earlier, we have reported a similar trend in alteration of α-helix of two homologous serum proteins in the presence of a subdomain IIA binder food dye tartrazine.37 It is reported in the literature that HSA is a stable and at the same time very flexible protein.72 The domains of HSA are capable to move substantially while keeping their secondary structure almost unchanged73 and that is why, in our case, the increase in α-helix content of HSA is found to be considerably less than other reports.8,9,74 The CD spectra have also been used to determine the nature of binding interaction of ligand with serum proteins.37 Increasing salt concentration leads to the lowering in strength of the electrostatic interaction, while the nonelectrostatic modes become prominent factor and consequently CD spectra can get modified with salt.37 Therefore, we have recorded the CD spectra of serum proteins in the presence of salt (0.2 M NaCl) at pH 7.4. In our experimental salt concentrations, the secondary structure of both serum proteins is not found to be altered, which has been confirmed by separate blank run. It has been observed that binding of C343-anion with BSA is affected by salt addition and is reflected in the CD spectra. Change in αhelical content of BSA due to binding with C343-anion is found to be 1.5 units (∼2.5% increase with respect to protein without ligand) in the absence of salt, while the same in the presence of salt is only 0.5 units (∼0.8% increase). The strength of complexation of BSA with C343-anion weakens with the addition of salt, whereas the same for the HSA:C343-anion complex is but little reduced. On the other hand, the CD spectra of the HSA:C314 complex is not at all perturbed with salt addition. These results justify our earlier observations that the primary mode of interaction of C343-anion with both the serum proteins is electrostatic and that of C314 with HSA is nonelectrostatic in nature.

Figure 10. Reorganization energy (RE) for C343-anion in varying concentrations of HSA and BSA in pH 7.4 at 300 K.

Figure 11. Reorganization energy (RE) for C314 in varying concentrations of HSA in pH 7.4 at 300 K.

From Figures 10 and 11, it is observed that with increasing concentration of the protein RE decreases and after certain concentration it becomes practically constant. The reorganization energies for HSA:C343-anion and BSA:C343-anion complexes at the point of saturation of interaction are found to be (1410 ± 3) and (1440 ± 5) cm−1, respectively, whereas the corresponding value for water:C343-anion system is found to be (1485 ± 4) cm−1. On the other hand, reorganization energy for the HSA:C314 complex is (2035 ± 8) cm−1 and the 6081



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corresponding value for water: C314 system is (2140 ± 10) cm−1. Thus, ∼80−100 units’ difference in reorganization energy (RE) between water:fluorophore and protein:fluorophore system indicates that the fluorophore is facing two different environments in those two situations; this means that with increasing concentration of proteins, fluorophore is distributed from water to the protein cavity. Similar order difference in reorganization energy between apomyoglobin:C343 and water:C343 systems have also been reported elsewhere.77 It was discussed there that due to enhanced solubility in water, C343 has low partition coefficient between protein and water compared with other coumarin dyes like coumarin 153. RE represents the amount of energy required when the surrounding solvent molecules around the polar fluorophore organize themselves in such a way that can stabilize the excitedstate dipole. In other words, it can also be conceived as the amount of energy required to disorganize the surrounding solvent molecules around a polar fluorophore from an organized state.30 With increasing concentration of protein, the fluorophore switches from a more polar bulk aqueous phase to the less polar pocket, which affects the solvent organization/ orientation in the bulk and the pocket as well, and is reflected in the change in the magnitude of reorganization energy. A certain concentration of protein beyond which the complexation of coumarin dyes is complete, there is no further shift in the emission maxima and almost a constant value of the calculated solvent reorganization energy is observed. RE at the point of saturation for the HSA:C343-anion complex is lower compared to the BSA:C343-anion complex. The solvent reorganization energy depends on the change of water structure while switching from the bulk aqueous phase to the protein environment. As the bound C343-anion in BSA is little exposed to water, the change in water structure should be less as compared to HSA and is the reason for showing comparatively low reorganization energy. The observations in the earlier sections (steady state and time-resolved anisotropy, REE, estimation of micropolarity, and microviscosity) reveal that the binding site of C343-anion is deep-rooted in the less polar cavity of subdomain IIA of HSA, while the effective binding pocket of C314 in HSA is more accessible to water. It is interesting to note that the change in RE while moving from bulk aqueous phase into HSA is higher for the HSA:C314 complex than for the HSA:C343-anion complex. We have discussed earlier in Time-Resolved Measurements that due to conformational readjustment HSA can accommodate the neutral C314 as well and thereby the perturbed binding pocket becomes more water accessible. From the literature report, it is clear that such local conformational alteration of individual domain does not lead to substantial change in global secondary structure of serum proteins.73 But in this respect, reorganization energy is an important parameter which can act as a descriptor of local water/solvent structure. Any slight change in local conformation of proteins can be associated with the change in solvent accessibility of amino acid side chains and peptide backbone. Water molecules in the binding cavities form hydrogen-bonded network with the interior residues and lead to the structural integrity of proteins78 and therefore any modification in local conformation leads to change in the hydrogen-bonding network, that is, change in water structure.78 Therefore, for a higher change in RE for the HSA:C314 system, it may be presumed that for an accommodative response to the perturbation by the guest, the host adopts some suitable

altered conformation from the initial equilibrium solution structure surmounting more RE. 3.9. Analysis of Docking Simulation. In order to find out the preferred location within the SA pocket, we have investigated the docking of C343-anion into the crystal structure of HSA33 and BSA17 using Auto Dock.79 The docking simulation results have been found to corroborate the experimental results very well. The energetically favorable docked pose of C343-anion in HSA showing nearby residues within 3.5 Å is depicted below (Figure 12a).

Figure 12. Interacting amino acid residues around the binding site of docked C343-anion in the subdomain IIA cavity of (a) HSA and (b) BSA.

From Figure 12a, it is observed that C343-anion docks at a distance of ∼3.5 Å from Trp 214 of HSA in subdomain IIA. The other amino acid residues present in the binding site within similar separations are Arg 218, Arg 222, Lys 195, Lys 436, Ala 191, Val 455, Asp 451, and Leu 198. The change in solvent accessible surface area (SASA) of Trp 214 and other nearby amino acid residues in HSA are found to be greater than 10 Å2 (shown in Table T3 of the Supporting Information) which indicates binding interaction. The lowest energy docked pose of C343-anion with BSA is shown above (Figure 12b). From the docked pose (Figure 12b), it is found that C343anion is also positioned at ∼3.5 Å distance from Trp 213. The other amino acids available within ∼3.5 Å radius from the ligand are Arg 194, Arg 198, Arg 217, Lys 294, Ser 343, Ser 453, Ala 341, Val 342, Tyr 340, Leu 197, and Asp 450. The change in the solvent accessible surface area of these amino acid residues can be found in the Table T3 of the Supporting Information. A considerable change (>10 Å2) in solvent accessible surface area (SASA) of the residues with positively charged side chains like Arg and Lys in both proteins reveals that the mode of interaction is electrostatic in nature. Importantly, the change in solvent accessible surface area by more than 10 Å2 for some other residues with uncharged side chains in the pocket can also be found for HSA but not for BSA (see Table T3 of the Supporting Information); this indicates that nonelectrostatic modes are also operative when C343anion binds the subdomain IIA cavity of HSA. 6082



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Figure S9: plot of ΔH0 versus TΔS0 for the HSA:C343-anion complex in pH 7.4 at 300 K. Figure S10: fluorescence decay profiles of C314 in the absence and presence of HSA in pH 7.4 at 300 K. λex = 408 nm and λem = emission maxima. [C314] = 1.5 μM. Figure S11: Stern−Volmer plot for dynamic quenching of C343-anion by HSA and BSA in pH 7.4 at 300 K. Figure S12: time-resolved fluorescence anisotropy decay of C314 in the absence and presence of various concentrations of HSA in pH 7.4 at 300 K. [C314] = 1.5 μM. Figure S13: CD spectra of HSA with increasing concentration of C314 in pH 7.4 at 300 K. [HSA] = 9 μM. Table T1: difference in amino acid sequence between HSA and BSA in subdomain IIA binding cavity. Table T2: the thermodynamic parameters associated with complexation of HSA with C314 at pH 7.4. Table T3: change in solvent accessible surface area (ΔSASA) in Å2 of interacting residues available in the binding site of C343-anion in HSA and BSA. This material is available free of charge via the Internet at http://pubs.acs.org.

CONCLUSION The present work shows how photophysical properties of two polarity-sensitive fluorophores, C343-anion and C314, are modified in the equivalent binding pocket of two model serum proteins. The photophysical behaviors of both the fluorophore are modified to different extents in these proteinous environments. This has been exploited to determine the nature of the microenvironment around fluorophore, that is, micropolarity and microviscosity in the binding pocket. The results suggest that quenching of C343-anion fluorescence by BSA is primarily due to ground-state electrostatic complexation, and that by HSA is due to the ground-state interaction guided by both electrostatic and nonelectrostatic interactions. Also the C343-anion binds stronger with HSA than BSA. The binding location of C343-anion in HSA is in the hydrophobic core of subdomain IIA, whereas in BSA, the probe is placed in the more polar outer part of subdomain IIA (i.e., a more solventexposed region of the pocket). REE reveals that water molecules penetrate deeper into the cavity of subdomain IIA of HSA and the mobility of water molecules in the immediate vicinity of fluorophore is greatly restricted. The individual response of two serum proteins to the perturbation by ligand structure is found to be quite different from one another. HSA binds both C343-anion and neutral C314, but BSA is able to bind only the C343-anion. Although the major drug binding site (subdomain IIA) in both proteins contains anion receptors (i.e., prefers to bind anionic ligands), but HSA is likely to adopt favorable conformation to accommodate neutral ligands as well. During such conformational readjustment, the water structure in the perturbed binding pocket is altered to a larger extent, which is reflected by the higher change in RE of the HSA:C314 complex. On the other hand, the solution structure of BSA is less flexible and, hence, is not so accommodative. The present study shows an approach in scrutinizing vividly the subdomain IIA of SAs with the help of optical spectroscopy, and the outcomes can be very useful in rational design of suitable drugs having specific selection of their receptor binding pocket. This study also reveals that the structural homology between two serum proteins does not guarantee their functional relationship. Therefore, BSA cannot be a simple substitute of HSA for future studies, and this may also be applicable to other homologous proteins.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +91-3222-283314. Fax: +91-3222-282252. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank DST SERB-India (Grant SB/S1/PC-041/2013) for financial support. S.D. thanks IIT Kharagpur for a fellowship. We thank Prof. N. Sarkar for help in measuring time-resolved data in his lab-setup, and we also thank Prof. S. Dasgupta for giving us the opportunity to measure steady-state anisotropy. S.D. thanks Mr. N. Mahapatra and Dr. P. Bolel for help in various instances. We would also like to thank anonymous reviewers for their critical comments and suggestions.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

Detailed methodology of molecular docking simulation. Figure S1: emission spectra of C343-anion in absence and presence of different concentrations of BSA in pH 7.4 at 300 K. Figure S2: emission spectra of C314 in the absence and presence of (a) HSA and (b) BSA in pH 7.4 at 300 K. Figure S3: emission spectra of BSA-bound C343 in the absence and presence of increasing concentration of warfarin (WF) in pH 7.4 at 300 K. Figure S4: emission spectra of C343-anion complexed with HSA with increasing NaCl concentration in pH 7.4 at 300 K. Figure S5: emission spectra of C314 complexed with HSA with increasing NaCl concentrations in pH 7.4 at 300 K. Figure S6: Job’s plot for the BSA−C343 complex in pH 7.4 at 300 K. The total concentration of C343 and BSA is 6 μM. Figure S7: plot of 1/f B versus [protein]−1 for the calculation of association constant (KAniso) of the HSA/BSA:C343-anion complex from steady-state anisotropy data. Figure S8: plot of ΔH0 versus TΔS0 for the BSA:C343-anion complex in pH 7.4 at 300 K. 6083



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