Hydrophobicity Is the Governing Factor in the Interaction of Human

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Hydrophobicity Is the Governing Factor in the Interaction of Human Serum Albumin with Bile Salts Narayani Ghosh, Ramakanta Mondal, and Saptarshi Mukherjee* Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Indore By-Pass Road, Bhauri, Bhopal 462 066, Madhya Pradesh, India S Supporting Information *

ABSTRACT: The present study demonstrates a detailed characterization of the interaction of a series of bile salts, sodium deoxycholate (NaDC), sodium cholate (NaC), and sodium taurocholate (NaTC), with a model transport protein, human serum albumin (HSA). Here, steady-state and timeresolved fluorescence spectroscopic techniques have been used to characterize the interaction of the bile salts with HSA. The binding isotherms constructed from steady-state fluorescence intensity measurements demonstrate that the interaction of the bile salts with HSA can be characterized by three distinct regions, which were also successfully reproduced from the significant variation of the emission wavelength (λem) of the intrinsic tryptophan (Trp) moiety of HSA. The time-resolved fluorescence decay behavior of the Trp residue of HSA was also found to corroborate the steady-state results. The effect of interaction with the bile salts on the native conformation of the protein has been explored in a circular dichroism (CD) study, which reveals a decrease in α-helicity of HSA induced by the bile salts. In accordance with this, the esterase activity of the protein−bile salt aggregates is found to be reduced in comparison to that of the native protein. Our results exclusively highlight the fact that it is the hydrophobic character of the bile salt that governs the extent of interaction with the protein. Isothermal titration calorimetry (ITC) and molecular docking studies further substantiate our other experimental findings.

1. INTRODUCTION Bile salts are naturally occurring amphiphilic molecules that are produced in the liver and released into the duodenum upon food intake and are known to play key roles in solubilization and the absorption of fats and other fat-soluble substances in the body.1,2 The aggregation behavior of bile salts is more complex in nature as compared to that of common surfactants forming micelles.3 To date, several methods have been used to interpret the self-organization behavior of bile salts. According to the most widely accepted model,4,5 the primary aggregates are formed at a low concentration of bile salt driven by the hydrophobic interaction of the convex surface of the monomers (usually 2−10 monomeric units). The hydrophilic groups are believed to be oriented outward in the primary aggregates.4,5 At higher concentrations, the primary aggregates agglomerate to form secondary aggregates having elongated rodlike morphology.4,5 The presence of different tunable binding sites makes bile salt aggregates an interesting host system capable of encapsulating various hydrophobic and hydrophilic guest molecules,1−5 which contributes significantly to the vast application of these supramolecular assemblies over a broad spectrum of biological and biotechnological fields. Bile salts have been used in several steps of protein purification, such as selective solubilization of membranes, chromatographic separation, and reconstitution of proteins.6−9 The efficiency of © 2014 American Chemical Society

protein purification by bile salts mainly depends on the nature of interaction of proteins with the bile salts. Therefore, a quantitative study of protein−bile salt interaction is required for an in-depth understanding and molecular-level interpretation of the bile salt-mediated protein purification method. Although the interaction between proteins and bile salts is scarcely documented, most of the studies reported so far are timeconsuming and tedious.10−14 Hence, this important field of study of protein−bile salt interaction remains open to further exploration. Bile salts form reversible complexes with serum proteins, which play an important role in the transport of these ligands from the blood to the liver.15 The polar substitutions (number, position, and type) on the steroid ring significantly govern the binding affinity of bile salts with proteins.16 Human serum albumin, HSA, a transport protein, consists of 585 amino acid residues and is characterized by a low tryptophan (Trp) and high cysteine content, stabilizing a series of nine loops.17 The secondary structure of native HSA is composed of ∼67% α-helix with six turns and 17 disulfide bridges, and the tertiary structure of HSA is composed of three domains, I−III, each of which is further subdivided into two subdomains, A and B.17,18 In spite of its complex structure, Received: October 29, 2014 Revised: December 29, 2014 Published: December 30, 2014 1095

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Langmuir HSA can be studied by monitoring the intrinsic fluorescence being rendered by the lone Trp214 amino acid residue. The serum albumins are known for their surprising ability to accommodate a broad range of exogeneous and endogeneous ligands including small amphiphilic molecules such as bile salts. In this study, we have deciphered the binding interaction of a series of bile salts having varying polarity (sodium deoxycholate, NaDC; sodium cholate, NaC; and sodium taurocholate, NaTC) with plasma protein HSA by means of fluorescence spectroscopy exploiting the remarkable sensitivity of the intrinsic fluorescence of HSA with respect to its immediate microenvironment.19,20 Our steady-state and time-resolved fluorescence spectroscopic data distinctly reveal the various stages of binding of the protein, HSA, with the bile salts as a function of the varying concentration of the latter. A thorough quantitative analysis of the data further shows a significant dependence of the interaction on the hydrophobicity of the bile salts employed. The thermodynamics of HSA−bile salt interaction has been rigorously explored with isothermal titration calorimetric (ITC) experiments, which leads to deeper insight into the molecular-level understanding of the interaction mechanism. The thermodynamic data show that the interaction is principally an entropy-driven process that is rationalized from the concept of the classical hydrophobic effect accompanying the release of solvating water molecules as a result of such interaction. Circular dichroism (CD) spectroscopy has been applied to study the influence of bile salts on the native protein conformation. Furthermore, the AutoDock-based blind docking simulation results are found to complement the experimental findings and establish the fact that the extent of interaction is a function of the hydrophobicity of the bile salt used. The effect of bile salt on the functionality of the protein has also been studied in terms of the esterase-like activity of HSA, which is found to vary in agreement with the effect on protein conformation.

F0 A = 1 + KSV [Q] F

(1)

where F0 and F are the fluorescence intensities of Trp214 in the absence and presence of acrylamide, respectively, and [Q] is the concentration of acrylamide. 2.2.3. Time-Resolved Fluorescence Measurements. Fluorescence lifetimes were recorded by the time correlated single-photon counting (TCSPC) technique. The samples were excited using an IBHNanoLED-295 (having a fwhm of IRF of ∼810 ps) light source, and the signals were collected using a Hamamatsu MCP photomultiplier (model R-3809U-50) at a magic angle polarization of 54.7°.19 The decays were deconvoluted with DAS-6 decay analysis software. 2.2.4. Circular Dichroism (CD) Measurements. The CD spectra were recorded on a JASCO J-815 spectropolarimeter using a cylindrical cuvette of 0.1 cm path length at 25 °C. Each CD spectrum is an average of four successive scans obtained at a 20 nm/min scan rate with an appropriately corrected baseline. 2.2.5. Isothermal Titration Calorimetry (ITC) Measurements. The ITC experiments were carried out on a Nano ITC (TA Instruments) at 25 °C. Thoroughly degassed solutions of both HSA and bile salts were loaded into the calorimeter cell to ensure that no bubbles were formed. A total of 25 aliquots of bile salt solutions (2 μL for each injection) were injected from a rotating syringe (300 rpm) into the ITC sample chamber containing the protein solution at an interval of 180 s between each injection. Corresponding control experiments to determine the heat of dilution of the bile salts were performed by injecting an identical volume of the same concentration of the bile salts into the buffer. The ITC data were analyzed using the NanoAnalyze v2.4.1 software supplied with the instrument according to a model for one set of independent binding sites. The heat associated with each bile salt−buffer mixture was subtracted from the corresponding heat of bile salt−protein reactions to obtain the actual heat of interaction. In one set of the independent binding sites model, all of the binding sites are considered to be identical, and no distinction between interacting sites is made. Here, each macromolecule is considered to comprise only one type of binding site with a finite number of identical noninteracting binding sites, all of which have the same intrinsic affinity for the ligand. This implies homogeneous binding between the ligand (here, the bile salt) and the macromolecule (here, HSA). We assume that n, the number of ligands binds per macromolecule with identical thermodynamics, the reaction stoichiometry (n), the binding association constant (Ka), and the change in enthalpy (ΔH) are determined by fitting the raw data. The simple case of a one-site model can be written in terms of the binding association constant given by Ka = [ML]/[M][L], where [ML] is the concentration of the protein−bile salt complex and [M] and [L] are the concentrations of the free macromolecule (here, HSA) and free ligand (here, bile salts), respectively. As the titration proceeds, [M], [L], and [ML] change constantly, but Ka remains constant. By simple algebraic manipulation of the equation mentioned elsewhere,21,22 an expression for Δq (change in raw heat/energy for the reaction) is obtained in terms of concentrations, ΔVcell (change in cell volume), ΔH (change in enthalpy), and Ka. The equation was then fit to the ITC data by optimizing Ka and ΔH to minimize squared sum of the errors between measured and calculated values of Δq. It is pertinent to mention in this context that according to the convention of the accompanying software with our ITC instrument the endothermic and exothermic heat burst curves are shown in downward and upward directions, respectively. However, it is important to note that the upward/downward directions of heat changes as exemplified by any ITC enthalpogram are not universal and depend on the instrument used rather the sign of ΔH as obtained from the fitting of the experimental data providing the genuine thermodynamic signature of the concerned process, and we have interpreted our data accordingly. 2.2.6. Esterase-like Activity Assay. The esterase-like activity of HSA has been exploited to investigate the effect of binding the bile salts on the functionality of the protein. Here, the action of HSA on pnitrophenyl acetate (PNPA, that is, the substrate) has been

2. EXPERIMENTAL SECTION 2.1. Materials. Human serum albumin (HSA), the bile salts (NaDC, NaC, and NaTC, Scheme S1 in the Supporting Information (SI)), and acrylamide were used as purchased from Sigma-Aldrich Chemical Co., USA. Tris buffer was obtained from Sigma-Aldrich Chemical Co., USA, and 0.01 M Tris-HCl buffer of pH 7.40 was prepared in deionized triply distilled Milli-Q water. The purity of the solvent was tested by running the fluorescence spectrum in the studied wavelength region. The concentration of HSA was 10 μM for the steady-state and time-resolved experiments, whereas it was 5 and 50 μM for CD spectroscopy and ITC experiments, respectively. 2.2. Instrumentation and Methods. 2.2.1. Steady-State Spectral Measurements. The absorption and fluorescence spectra were recorded on a Cary 100 UV−vis spectrophotometer and a Fluorolog 3-111 fluorometer, respectively, using freshly prepared solutions. The intrinsic fluorescence of the protein (HSA) was recorded by exciting the protein samples at 295 nm in order to minimize the contribution from the tyrosine amino acid residues present in HSA.17−20 2.2.2. Acrylamide Quenching. Acrylamide was used to quench the Trp214 fluorescence in HSA in the presence of different concentrations of NaDC. Acrylamide is known for its activity as an efficient quencher of tryptophan fluorescence of serum albumins.19 The quenching experiments were performed by adding measured volumes of aliquots from a 4 M acrylamide stock solution to a protein solution (10 μM) to achieve the desired acrylamide concentration. The excitation wavelength was 295 nm. The Stern−Volmer quenching constants (KASV) in the presence of acrylamide were then calculated with the following equation19 1096

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Figure 1. Steady-state fluorescence spectra of HSA in the presence of varying concentrations of (A) NaDC (1 → 10:0, 0.05, 0.08, 0.3, 0.6, 1.0, 1.5, 2.5, 3.5, 7 mM NaDC), (B) NaC (1 → 9:0, 0.1, 0.8, 2, 4, 6, 10, 15, 30 mM), and (C) NaTC (1 → 9:0, 0.1, 0.8, 2, 4, 6, 10, 15, 27 mM). [HSA] = 10 μM.

ligands and/or drug molecules.17,18,20,29−32 In the present case, upon interaction of bile salts with HSA the fluorescence intensity is found to undergo prominent quenching along with a discernible blue shift in emission wavelength (∼17 nm for NaDC and ∼13 nm for both NaC and NaTC) as displayed in Figure 1. This observation can be attributed to the formation of the protein−bile salt complex, which results in Trp being buried in the hydrophobic cavities within the protein scaffolds, and our results are in excellent agreement with other literature reports.20,32 This finding can be described on the basis of two assumptions: (i) the internalization of fluorophore (Trp) toward the core of the protein and (ii) bile salt-induced unfolding of the protein structure, which consequently leads to a more hydrophobic interaction between the hydrophobic moieties of the bile salt and the fluorophore (Trp). The blue shift in emission maxima in the presence of bile salts suggests that Trp214 experiences a more hydrophobic environment as compared to that in the absence of bile salts. Hence, we performed acrylamide quenching studies and monitored the accessibility of Trp214 with respect to acrylamide (Figure S1, SI). As seen in the figure, the KASV values decrease with an increasing concentration of NaDC, signifying that Trp214 becomes less accessible toward acrylamide, and this can happen only when Trp214 becomes more deeply seated and buried inside the protein scaffolds. The binding isotherm for the HSA−bile salt interaction has been constructed from the variation in the fluorescence spectral properties making use of the fraction of protein bound to the bile salts (α) as given by

investigated by monitoring the absorbance of the released product, pnitrophenol, on a Cary-100 UV−vis spectrophotometer (λabs = 400 nm, molar absorption coefficient ε = 17 700 M−1 cm−1).23,24 Briefly, the reaction conditions were maintained as [HSA] = 25.0 μM, [PNPA] = 50.0 μM, pH 7.40, and temperature = 37 °C (the temperature was kept constant at the given value by a recycling flow of water, with an accuracy of up to ±0.5 °C). It is noteworthy in this context that the bile salts exhibit only negligible absorbance at the monitoring wavelength of 400 nm, which consequently discards any possible contamination due to any optical screening effect originating from light absorption by bile salts. 2.3. Molecular Docking Studies. The docking studies were performed with the AutoDock 4.2 software package that utilizes the Lamarckian genetic algorithm (LGA). The native structure of HSA was taken from the Protein Data Bank and has a PDB entry of 1AO6.25 The 3D structures of the bile salts were prepared on AutoDock 4.226 from the optimized geometries at B3LYP/6-31G as obtained from Gaussian 03W.27 The grid size was set to 126, 126, and 126 along the X, Y, and Z axie with a 0.375 Å grid spacing. The AutoDocking parameters were GA population size = 150, maximum number of energy evaluations = 250 000, and GA crossover mode = two points. The lowest-binding-energy conformer was searched out of 10 different conformations for each docking simulation and used for further analysis. The PyMOL software package was used for the visualization of the docked conformations.28

3. RESULTS AND DISCUSSION 3.1. Deciphering the Protein−Bile Salt Interaction Using the Intrinsic Fluorescence of HSA. Upon addition of the bile salts, the absorption spectra of HSA remain almost unaltered, whereas more dramatic changes were observed in the emission profiles. The emission maximum of HSA in aqueous buffer was centered at ∼348 nm, which indicates that the Trp residue is embedded in a hydrophobic interior of the native protein.17−20,29−32 Because the intrinsic fluorescence of HSA is known to be sensitive to its microenvironment,17−20,29−32 it can be used to probe the interaction with a host of exogenous

α=

Iobs − I0 Imax − I0

(2)

where I0, Iobs, and Imax represent the fluorescence intensities of Trp in the absence of bile salt, at an intermediate concentration 1097

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Figure 2. Binding isotherm for the HSA−bile salt interaction present in the form of α vs [bile salt]. (A) NaDC (●), (B) NaC (○), and (C) NaTC (■). Insets show the variation of the fluorescence intensity of HSA with varying bile salt concentrations. [HSA] = 10 μM.

bound to the bile salt attains saturation beyond C2. The extent of binding of the bile salts to HSA can be quantitatively accessed from the data as presented in Figure 2, which reveals an ∼4% decrease in the Trp fluorescence intensity in region I (up to C1) following the interaction of HSA with NaDC, whereas the extent of change is remarkably enhanced (∼25%) in region II, the co-operative binding zone (up to C2), which is followed by saturation. The pattern of binding of the other two bile salts, namely, NaC and NaTC, is also similar to that of NaDC, but the extent of binding is different. From the absolute values of C1 and C2 (as mentioned in Table S1 in SI), it can be rationally concluded that the extent of binding is maximized for NaDC and follows the order NaDC > NaC > NaTC, which is a clear signature of the fact that it is the degree of hydrophobicity of the bile salts that is the driving force for quantifying the interaction of the bile salts with the protein. The presence of an additional hydroxyl moiety in the steroid ring of NaC makes it less hydrophobic than NaDC, and the presence of a hydrophilic sulfonate group in NaTC contributes significantly to increasing its hydrophilicity.1−5,10−14 Therefore, the affinity of HSA for bile salts decreases with increasing polarity (decreasing hydrophobicity) of the bile salts. This argument has been further substantiated from the study of the associated thermodynamic parameters of the protein−bile salt interaction, as discussed in a forthcoming section. From our acrylamide quenching studies, the magnitudes of KASV do not undergo any appreciable change beyond ∼2 mM NaDC, which happens to be very close to the break point, C2,

of the added bile salt, and at the saturation level of interaction, respectively.33 The binding isotherms for the interaction of all three bile salts with HSA are depicted in Figure 2, which exhibits two breakpoints denoted by C1 and C2. Figure 2 distinctly reveals that the binding of bile salts to HSA takes place in distinct stages and in a sequential manner. In the native state, the protein inherently has some highly energetic sites that are readily available to accommodate the bile salts. These sites are extremely active in nature, and hence at low concentrations the binding of bile salt(s) to HSA is highly specific. By analogy to literature reports describing the interaction of serum albumins with other surface-active agents,31,32 the initial decrease in fluorescence intensity (insets of Figure 2) up to C1 (region I) can be attributed to the change in microenvironment in the vicinity of the intrinsic Trp residue of HSA as bile salts bind to the highly energetic sites of the protein.32 Consequently, the protein opens up and loses some of its secondary structure (discussed in detail in section 3.3), thereby enabling the protein to bind to the subsequently added bile salts in a cooperative manner. This is exemplified by the decrease in fluorescence intensity (insets of Figure 2) and a sharp increase in the fraction (α) of HSA bound to the bile salts (Figure 2). After C2, representing region III in the insets of Figure 2, the change in emission intensity is not that significant, which indicates that beyond C2 the protein is almost saturated with the bile salts and further binding may not be consequential. This observation is well supported by Figure 2, which also corroborates the fact that the fraction (α) of protein 1098

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Figure 3. Variation of emission wavelength maxima of HSA with increasing concentrations of (A) NaDC (●), (B) NaC (○), and (C) NaTC (■). [HSA] = 10 μM.

Figure 4. Time-resolved fluorescence decay curves of HSA with increasing concentrations of (A) NaDC, (B) NaC, and (C) NaTC. [HSA] = 10 μM.

bile salt binding isotherms for all three bile salts under investigation. As seen in Figure 3, the plot of the variation of λem with bile salt concentration also reveals that the interactions are sequential in nature. This specific pattern of observation can be rationalized from similar arguments as stated earlier. It is noted that the concentrations of the bile salts required to impart the same change in protein conformation vary in the order NaTC > NaC > NaDC, which can be attributed to the

in Figure 2. This again proves that in all probability the microenvironment in and around Trp214 remains almost uniform beyond C2, as described later. It is important to note in this context that the emission wavelength of the intrinsic Trp moiety of HSA also undergoes a specific variation following interaction with the bile salts, as can be seen from the emission spectra presented in Figure 1. Figure 3 shows that the variation of emission wavelength maxima (λem) with increasing concentration of bile salts reasonably reproduces the HSA− 1099

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Figure 5. Variation of the average lifetime of HSA with increasing concentrations of (A) NaDC (●), (B) NaC (○), and (C) NaTC (■). [HSA] = 10 μM.

The typical time-resolved fluorescence decay profiles of the intrinsic fluorescence of HSA in the presence of various concentrations of bile salts are displayed in Figure 4, and the decay parameters are compiled in Table S2 in SI. It is clearly seen in Figure 4 that the lifetime of Trp in HSA gradually decreases upon addition of the bile salts. The stepwise binding of the bile salts with HSA is further established from the variation of the average fluorescence lifetimes (⟨τ⟩) of Trp (Figure 5). Here also, the pattern of variation of the average lifetimes of Trp upon interaction with the different bile salts follows a trend similar to that encountered in the steady-state measurements. From the variation of the average lifetime values of Trp as depicted in Figure 5, it is clear that the binding of the bile salts occurs in distinct stages and the corresponding concentrations of the break points of Figure 5 are in excellent agreement with those obtained from our steady-state results (Table S1 in SI). The reduction in the average lifetimes of the Trp moiety of HSA can be ascribed to the combined effect of quenching and the conformational transitions in Trp induced by the added bile salts.32,35,36 As seen in Figure 5, for all three bile salts, the nature of binding to HSA is similar (characterized by two break points, C1 and C2); however, the extent of binding depends upon the bile salt used. By analogy to the steady-state results, the first break point (C1) corresponds to that concentration of the bile salt up to which the highly energetic sites inside the protein scaffolds are available. Beyond C1, the protein undergoes bile salt-induced conformational changes that enable further binding of the bile salt to the protein in a cooperative manner, and this addition continues until concentration C2 is reached, beyond which a saturation phase is encountered. The role of the hydrophobicity of the bile salts is also highlighted in the fluorescence lifetime experiments; the binding follows the order NaDC > NaC > NaTC, and the reason for this variation has already been discussed in section 3.1.

decreasing order of hydrophobicity as stated earlier (Table S1 in SI). 3.2. Fluorescence Lifetime Measurements. In aqueous buffer, HSA exhibits a biexponential decay that is explained on the basis of the rotameric model of Trp.19,32,34,35 The three possible conformers of Trp are as depicted below:19,32,34,35

Of the biexponential decay pattern of Trp in native HSA, the faster component is ascribed to conformer C, and the slower component is argued to originate from the rapidly interconverting A and B conformers. However, the conversion of stable conformer C to A or B is believed to be difficult on the nanosecond time scale.34 Thus, a legitimate estimate of the relative population of the different rotamers can be obtained from the contributions of their characteristic lifetimes.36 The conformation of the indole ring of Trp is slightly puckered in the ground state as here the p(N) orbital overlap with the rest of the π system is slightly compromised. This leads to a decrease in the delocalization of electrons through the indole ring. The later argument has also been substantiated by Alcala and coworkers.36 However, the conformation attains planarity following photoexcitation to the excited state presumably because of delocalization of the nitrogen lone pairs involving the aromatic system.36 It is usually believed that interaction with quencher molecules perturbs the planarity of the indole ring.32 This results in changing the microenvironment in the vicinity of Trp, leading to a decrease in the fluorescence lifetime.32,36 1100

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Figure 6. Far-UV CD spectra of HSA in the presence of various bile salt aggregates (A) NaDC (1 → 8:0, 0.03, 0.33, 1.33, 2.66, 5.33, 6.66, 8 mM), (B) NaC (1 → 5:0, 0.53, 18.6, 23, 25.5 mM), and (C) NaTC (1 → 5:0, 0.53, 18.6, 23, 25.5 mM). [HSA] = 10 μM.

A quantitative analysis of the far-UV CD spectra provides a deeper understanding of the nature of the interaction with bile salts in the secondary structure of HSA. First, the mean residue ellipticity (MRE) is calculated from the recorded ellipticity (θobs in mdeg at 222 nm) value according to the following equation:32,38,39

The relative contributions of lifetimes (Table S2 in SI) suggest that the total intensity decay of Trp in the absence of any bile salt is composed of a faster component (amplitude ∼18%) and a slower component (amplitude ∼82%). The former can be attributed to the contribution from conformer C, and the latter is from conformer(s) A and/or B.32,34 The modulation in the relative amplitudes (α1 and α2, Table S2) of the lifetime components upon interaction with the bile salts is noteworthy. For example, at the saturation level of the HSA− NaDC interaction ([NaDC] ≈ 7 mM), the contribution from conformer C increases to ∼38% along with a concomitant decrease in the contribution of conformer A and/or B to ∼62%. In the native conformation of HSA, the Trp214 residue remains buried in a constrained hydrophobic environment19,37−39 and thereby resists a facile interconversion of conformer C to A or B.32,35,36 At the saturation level of the protein−bile salt interaction, the protein undergoes a loss of its secondary structure, resulting in the loss of its rigidity. Therefore, the possibility of the interconversion from C to A or B is augmented, as exemplified by the lowering of the amplitude of the longer lifetime (α2 in Table S2). The rotation of the indole ring of the Trp moiety should be facilitated in the excited state because of the enhanced planarity of the ring. This is likely to contribute to increasing the probability of interconversion of the Trp rotamer to more stable conformer C as revealed through the increasing amplitude of the shorter component (α1 in Table S2).32,36 It is argued that the nonradiative decay routes in Trp214, mainly due to charge transfer from the indole ring to nearby substitutions, are accelerated, resulting in a decrease in lifetime (Figure 5, Table S2).32,34,35 3.3. Circular Dichroism (CD) Spectral Study. The far-UV CD profile of native HSA (Figure 6) exhibits two minima at ∼208 and ∼222 nm characteristic of the α-helix-rich secondary structure.19,31,32,37−39 The CD signal of HSA is found to decrease at all wavelengths with added bile salts, implying an induced perturbation of the secondary structure of the protein.

MRE(deg· cm 2· dmol−1) =

θobs c pnl × 10

(3)

Here, cp is the molar concentration of the protein, n is the number of amino acid residues (585 for HSA17−20,25,29), and l is the cell path length (here 0.1 cm). The α-helical content of the protein is then determined as29,32 %α − helix =

−(MRE − 2340) × 100 30 300

(4)

For native HSA in aqueous buffer (pH 7.40 at 25 °C), the αhelicity was estimated to be ∼66%, which is in good agreement with literature reports.17−20,29,30,38 Upon subsequent addition of NaDC to HSA, the α-helicity was gradually found to decrease, as is displayed in Table S3 in the SI. However, even from the CD results, we have proven that the structural loss of HSA upon binding with the bile salts is not that significant, especially for NaC and NaTC. For NaDC at around 7 mM, the α-helical content of HSA is reduced by ∼5%, thereby suggesting that among the three bile salts used in this study NaDC induces the maximum structural loss in the protein, which can be attributed to the greater hydrophobic character of NaDC as compared to that of the other two bile salts. This observation further suggests that the interaction of bile salts with HSA is principally hydrophobic in nature and bears a strong resemblance to our other experimental results. As seen in Table S3, the percentage loss of α-helix in NaDC is only ∼5%, whereas there is a far greater decrement (∼35%) of fluorescence intensity of Trp214 under saturating conditions 1101

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Langmuir of NaDC (∼7 mM). The decrease in Trp214 fluorescence intensity is mainly due to the loss of the secondary structure of HSA induced by NaDC. It must be stated here that CD spectroscopy is a “global reporter”; it gives us an estimate of the overall structural changes the protein undergoes as a result of the interaction with the different bile salts. However, steadystate fluorescence exclusively probes the microenvironment in and around the Trp214 amino acid residue, and hence it serves as a “local reporter.” Thus, it will not be rational to draw a direction correlation between the steady-state measurements and CD spectroscopy data although both of these techniques unambiguously support that the binding of bile salts induces structural perturbations of the protein. 3.4. Isothermal Titration Calorimetric (ITC) Study. To gain deeper insight into the mechanism of interaction between HSA and bile salts, the thermodynamic parameters have been evaluated using an Isothermal Titration Calorimetric (ITC) technique. ITC is a powerful tool for studying the thermodynamics of a host of biomolecular interactions (estimation of enthalpy change (ΔH), entropy change (ΔS) for the process under investigation at a given temperature).40−43 Thus, a detailed calorimetric study of the interaction of the three bile salts (NaDC, NaC, and NaTC) with HSA has been carried out. Figure 7 displays the

Table 1. Summary of the Thermodynamic Parameters for the HSA−Bile Salt Interaction as Obtained from ITC Measurements ΔH (kJ mol−1)

ΔS (J mol−1 K−1)

ΔG (kJ mol−1)

(7.85 ± 0.2) × 103

3.14 ± 0.50

85.10

−22.22

2

(7.39 ± 0.2) × 10

2.02 ± 0.45

61.67

−16.36

(3.64 ± 0.2) × 102

3.55 ± 0.56

60.93

−14.61

system

Ka (M−1)

HSANaDC HSANaC HSANaTC

of the fact that the hydrophobicity of the bile salts controls their binding to HSA. Our ITC results thus substantiate the steadystate and time-resolved data as well. From the thermodynamic parameters, it can be concluded that the binding interaction of all three bile salts with HSA is entropically favorable (ΔS > 0) but enthalpically unfavorable (ΔH > 0). An overall negative free energy of binding (ΔG < 0) reflects that the interaction of HSA with all three bile salts is spontaneous in nature. Such entropy-driven interactions are generally rationalized on the basis of the classical hydrophobic effect (release of solvating water molecules as a result of the interaction).45,46 Thus, the positive enthalpy change along with a positive entropy change establishes the predominance of the hydrophobic effect underlying the HSA−bile salt interaction.44 It is seen from Table 1 that the entropy change for the HSA−bile salts interaction process varies proportionately with increasing hydrophobicity of the bile salts (NaDC > NaC > NaTC). The free-energy change (ΔG) reflecting the spontaneity of the interaction also follows a similar trend, which further underlines the fact that the interaction is primarily hydrophobic in nature, and our ITC data strongly substantiates our spectroscopic results as discussed in foregoing sections. We have also performed a molecular docking study that shows that hydrophobic subdomain IIA is a favorable binding site for all three bile salts within the protein. It is noted that ΔGdock varies as NaDC (ΔGdock = −29.11 kJ mol−1) < NaC (ΔGdock = −22.21 kJ mol−1) < NaTC (ΔGdock = −18.24 kJ mol−1), which successfully corroborates the other experimental findings (details in Figure S3, SI). 3.5. Effect of Interaction of Bile Salts on the Activity of HSA. The effect of the bile salts under investigation on the activity of HSA has been evaluated in detail by monitoring the well-established esterase-like activity of HSA.23,24 Here, the action of HSA on the p-nitrophenyl acetate (PNPA) substrate is studied by monitoring the absorbance of the liberated product, p-nitrophenol (λabs = 400 nm). The details of the reaction conditions are mentioned in section 2.2. The influence of the interaction of the bile salts on the activity of HSA is shown in Figure 8 in terms of the relative modulation of the esterase-like activity of HSA in the presence of the bile salts in comparison to a control that is monitored for HSA alone in aqueous buffer. Here, one unit of activity has been defined as the amount of enzyme (HSA) required to liberate 1 μM of pnitrophenol from the substrate (PNPA) per minute at 37 °C. The observed reduction in the esterase-like activity of HSA following interaction with the bile salts is in keeping with the perturbation of the native protein conformation as discussed in previous sections.23,24 Also, it is interesting that the reduction of the activity of HSA follows the trend NaDC > NaC > NaTC, which is consistent with the extent of the loss of the native

Figure 7. ITC profile for the titration of HSA with NaDC at 25 °C in 0.01 M Tris-HCl buffer (pH 7.40). Top panel: Raw data for the integrated heat after the correction of the heat of dilution. Bottom panel: ITC enthalpograms obtained at 25 °C. The solid line is obtained upon fitting the data to a single set of sites binding model. See the Instrumentation and Methods section for the power sign convention.

representative ITC data for the interaction of NaDC in HSA at 25 °C. (The ITC profiles for the interaction of NaC and NaTC with HSA are given in Figure S2 of the SI.) The relevant thermodynamic parameters for the interaction of HSA with all of the bile salts under investigation as obtained from the fitting of the experimental ITC according to a model for one set of independent binding sites (Figure 7, Figure S2) have been summarized in Table 1. According to Ross et al., the nature of the underlying forces in a given interaction process has been briefly summarized as44 (i) ΔH > 0, ΔS > 0, corresponding to a hydrophobic effect; (ii) ΔH < 0, ΔS < 0, correspond to a van der Waals interaction, hydrogen bond formation; and (iii) ΔH < 0, ΔS > 0, corresponding to electrostatic/ionic interactions. As seen from the magnitudes of Ka (Table 1), the binding is an order of magnitude stronger for NaDC than for NaC and NaTC, whereas it is weakest for NaTC. This is a clear signature 1102

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Figure 8. (A) Modulation in relative esterase-like activity of HSA as a result of interaction with the bile salts. The activity is monitored by following the release of p-nitrophenol from the action of HSA and HSA−bile salt aggregates on p-nitrophenyl acetate (PNPA) (I, free HSA; II, HSA-NaTC complex; III, HSA-NaC complex; IV, HSA-NaDC complex). (B) Representative kinetic profiles for the release of p-nitrophenol from the action of HSA and HSA−bile salt aggregates on p-nitrophenyl acetate as indicated in the legend. [HSA] = 25.0 μM and [PNPA] = 50.0 μM.

quenching experiments. This work is dedicated to Professor Kankan Bhattacharyya on the account of his 60th birthday.

protein conformation upon interaction with these bile salts (as demonstrated in the CD spectral results in section 3.3).

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4. CONCLUSIONS The various spectroscopic techniques used herein conclusively establish the fact that the hydrophobicity of the bile salts seems to be the principal factor in governing the interaction of the bile salts with HSA. The binding of the three bile salts to the protein occurs in a sequential manner, and it is found that the extent of binding follows the order NaDC > NaC > NaTC, with NaDC being the most hydrophobic in nature. Molecular docking studies reveal that the bile salts bind in the IIA cavity of HSA and that this binding is spontaneous. Steady-state and time-resolved spectroscopic techniques corroborate each other very well, and from these results, the importance of the hydrophobic character of the bile salts has been substantiated. The interaction of HSA with bile salts is found to accompany the conformational perturbation of the native protein, and hence a commensurate influence on the activity of the protein (esterase-like activity) has been obtained. The ITC experiments establish that the interaction is mainly controlled by a hydrophobic effect showing the typical signature of an entropy-driven process that is interpreted from the concept of the classical hydrophobic effect.

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ASSOCIATED CONTENT

S Supporting Information *

Chemical structures of bile salts, acrylamide quenching, binding regions, fluorescence decay parameters, % α-helicity, ITC profiles, and a molecular docking study. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.G. acknowledges a research fellowship from the Government of India through CSIR-NET. R.M. acknowledges IISER Bhopal for a research fellowship. S.M. sincerely thanks DST, Government of India for financial support and Dr. Vishal Rai for providing some valuable suggestions. We also thank Dr. Bijan Kumar Paul for many stimulating discussions and Mr. Nirmal Kumar Das for helping us with the acrylamide 1103

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