Energetics of albumin-disuccinylmaslinic acid binding determined by fluorescence spectroscopy

Fluid Phase Equilibria 400 (2015) 43–52

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Fluid Phase Equilibria j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / fl u i d

Energetics of albumin-disuccinylmaslinic acid binding determined by fluorescence spectroscopy J.A. Molina-Bolívar a, * , C. Carnero Ruiz a , F. Galisteo-González b , M. Medina -ODonnell c, A. Parra c a b c

Departamento de Física Aplicada II, Escuela Politécnica Superior, Universidad de Málaga, Campus de Teatinos, 29071 Málaga, Spain Departamento de Física Aplicada, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071 Granada, Spain Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071 Granada, Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 February 2015 Received in revised form 4 May 2015 Accepted 7 May 2015 Available online 11 May 2015

Interaction of disuccinylmaslinic acid (SMA) with bovine serum albumin (BSA) has been investigated by fluorescence spectroscopic methods under different experimental conditions. From the temperature dependence of the binding process an extensive analysis of thermodynamic parameters has been made in connection with the drug structure. SMA binds to BSA mainly through electrostatic interactions at physiological pH (7.4) and low ionic strength. An increased electrolyte concentration provoked hydrogen bonds and van der Waals forces to control the complex formation. When pH was higher than the isoelectric point of albumin (i.e.p. 4.9) the attachment of the drug was favored by both negative enthalpy and positive entropy changes. These results suggest a dominance of electrostatic forces in the association process. Conversely, at pH values lower than i.e.p. the unfavorable negative entropy changes prompt the involvement of hydrogen bonds in the binding. A noteworthy enthalpy–entropy compensation phenomenon has been detected. The binding processes controlled mainly by hydrogen bonds and van der Waals interactions (pH 2.8, 4.2, and 7.4 at high NaCl concentration) fall in the same compensation line. The observation of this entropy-enthalpy compensation suggests that water reorganization plays an important role in the binding of SMA to BSA. Heat-capacity change (DCp) has been deduced from temperature dependence of enthalpy. Minimal values of DCp were found when electrostatic forces controlled the SMA–BSA association. The formation of a drug–albumin complex has been corroborated by electrophoretic mobility measurements. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Quenching fluorescence Thermodynamic parameters Enthalpy–entropy compensation

Introduction The binding of drugs to carrier proteins, such as serum albumins, is currently an active area of research because an understanding of the interaction mechanisms is crucial in order to understand the pharmacological activity of a certain drug [1–3]. In fact, from a pharmaceutical standpoint, it is widely accepted that the distribution, metabolism, and efficiency of many drugs can be controlled on the basis of their affinity for serum albumin [4]. On the other hand, some promising new drugs have proven ineffective because of their unusually high affinity for these types of proteins [5]. Consequently, the study of the interactions of drugs and serum albumins has important meaning in elucidating the transport and metabolism process of drugs, as well as in characterizing the fundamental interactions involved in the binding between

* Corresponding author. Tel.: +34 655602126. E-mail address: [email protected] (J.A. Molina-Bolívar). http://dx.doi.org/10.1016/j.fluid.2015.05.011 0378-3812/ ã 2015 Elsevier B.V. All rights reserved.

biomacromolecules and small molecules. Therefore, understanding the features of drug–albumin interactions can provide insights on drug therapy and design. One of the key aspects of such forces is the relationship between molecular structure and association thermodynamics. It is highly desirable that extensive and accessible thermodynamic characterization of drug–protein binding in relation to drug structure was available. Triterpenoids, abundant polycyclic compounds in the plant kingdom, can be found both in free form and as esters and glycosidic conjugates called saponins [6]. Although they were long considered biologically ineffective, recent evidence points to the beneficial effects both of naturally occurring triterpenoids as well as of synthetic derivatives against several types of human diseases, including certain cancers [7–10]. Naturally occurring triterpenoids such as oleanolic, betulinic, ursolic and maslinic acids have shown significant pharmacological activity, and recent studies suggest a promising potential of these products as anticancer agents [8–12]. Also, several studies examine their interaction with serum albumins [9,13–19]. Moreover, it has recently been observed that

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small structural modifications in these natural substances can produce advantageous derivatives with favorable chemotherapeutic properties [12,16,20]. In this context, we have focused on disuccinylmaslinic acid (cf. Chart 1 for molecular structure), which has recently been investigated, together with other derivatives of maslinic acid, regarding their capabilities as chemopreventive and chemotherapeutic agents [12,20]. However, beyond the potential applications of this substance as therapeutic compound, its molecular structure makes it an ideal candidate to examine the effect of pH and ionic strength on the binding mechanism to serum albumin, which is the main aim of the present work. Here, we have chosen bovine serum albumin (BSA) as the transport protein due to its water solubility, stability, and versatile binding capability, making it a model protein and a reference in both clinical and biochemistry assays [21]. In relation to spectroscopy, BSA contains two tryptophans (Trp), Trp134 near the surface of the protein in a polar environment, and Trp214 located in a hydrophobic pocket in the subdomain IIA, the so-called Sudlow I binding site [18]. These Trp residues are the source of its intrinsic fluorescence, which is very sensitive to microenvironmental changes brought about by the binding of different kind of molecules. In addition, fluorescence techniques are well known for their high sensitivity, reproducibility, and relatively easy use, making them widely applied to investigate the interaction between albumin and a substrate [22]. In this work, we have focused on the effect of both ionic strength and pH on the binding of SMA to BSA, proceeding as follows. First, we examine the fluorescence quenching of BSA produced by SMA at three different values of ionic strength at a fixed pH of 7.4 in a temperature range from 298.15 to 310.15 K. Second, maintaining a constant ionic strength (2 mM), we repeat the study at five different pHs in the same temperature range. From the analysis of the quenching data, we establish the binding constants at each ionic strength, pH, and temperature and then estimate the thermodynamic parameters for the drug–protein binding process under all the study conditions. The thermodynamic analyses are discussed in terms of the basic interactions acting between protein and substrate in each case. The data reported here could be useful not only to understand the binding mechanism between triterpenoid-type drugs and albumins, but also to design new synthetic drugs based on similar structural configurations. 1. Materials and methods 1.1. Materials Table 1 lists the main characteristics of the compounds used in this study. Due to the high purity grade of the commercial samples,

[(Chart_1)TD$FIG]

Chart 1. Molecular structure of disuccinylmaslinic acid.

they were used without further purification. Stock solutions of BSA were prepared in aqueous buffer solutions on the basis of its molecular weight (66,463) and kept in a refrigerator at around 4
C. A stock solution of disuccinylmaslinic acid (SMA), which was semisynthesized as described below, was prepared in ethanol. Working solutions with a fixed BSA concentration (25 mM) and varying SMA concentrations, ranging from 15 to 133 mM, were prepared daily, mixed thoroughly, and stabilized for at least 30 min at each temperature before the spectroscopic measurements. The ultra-pure water (resistivity 18 MV cm) used to prepare all the solutions was obtained by passing pure water from a Millipore Elix system through an ultra-high quality Millipore synergy purification system. All other reagents and solvents used in synthesis and analysis were of analytical reagent grade. 1.2. Semisynthesis of 2a,3b-2,3-Di-O-succinylmaslinic acid Commercially available reagents from Sigma–Aldrich (Madrid, Spain) were used without further purification: anhydrous pyridine (SKU number 270,970, 99.8%), succinic anhydride (SKU number 239,690, 99%) and triethylamine (SKU number T0886, 99%). Merck silica-gel 60 aluminium sheets (ref. 1.16835) were used for TLC. Merck silica-gel 60 (0.040–0.063 mm, ref. 1.09385) was used for flash chromatography. Analytical-grade dichloromethane (ref. D/1852/17) and acetone (ref. A/0600/17) from Fisher Scientific (Madrid, Spain) were used as eluents. The precursor maslinic acid was derived from olive pomace by the patented method by Garcia-Granados [23] and purified by a flash-column chromatography with silica gel as solid support and a mixture of dichloromethane/acetone as solvent. Succinylation of maslinic acid (472 mg, 1 mmol) was performed in dry pyridine (5 mL) with succinic anhydride (400 mg, 4 mmol) and triethylamine (0.38 mL, 4 mmol) overnight at 323  1 K followed by aqueous workup and flash-column chromatography with silica gel as a solid support and a mixture of dichloromethane/ acetone as a solvent. Thus pure disuccinylmaslinic acid was obtained at 90% yield. Mp 113–115
C; [a]D = +6
(c 1, MeOH); IR (KBr): nmax = 3346, 3021, 2803, 1688 cm1; dH (Py-d5) 5.50 (ddd, 1H, J1 = 4.6, J2 = 10.9, J3 = 11.4 Hz), 5.44 (dd, 1H, J1 = J2 = 3.5 Hz), 5.15 (d, 1H, J = 10.9 Hz), 3.30 (dd, 1H, J1 = 4.6, J2 = 14.0 Hz), 2.80–2.55 (m, 8H), 1.29, 1.25, 1.04, 1.01, 0.98, 0.96, 0.95 (s, 3H each); dC (Py-d5) 180.9, 176.0, 175.7, 173.5, 173.4, 145.7, 122.5, 81.2, 70.7, 55.5, 48.2, 47.2, 47.0, 44.6, 42.7, 42.4, 40.5 (2C), 39.2, 34.7, 33.8, 33.6, 33.3, 31.8, 31.0, 30.9, 30.6, 30.5, 29.0, 28.7, 26.7, 24.3, 24.2, 24.1, 18.9, 18.4, 17.7, 16.9; ESI-HRMS m/z calcd. for C38H57O10 [M + 1] 673.3947, found 673.3952. 1.3. Fluorescence measurements Steady-state fluorescence spectra were recorded using a FluoroMax-4 (Horiba, Jobin Yvon) spectrofluorometer, equipped with a 150-W xenon lamp and a cell housing with 1.0-cm pathlength quartz cuvettes, which was thermostated by a peltier unit. The emission spectra were collected from 300 to 500 nm with an integration time of 0.1 s using an excitation wavelength of 295 nm and slit widths for excitation and emission of 2 and 4 nm, respectively. At these wavelengths, a solution of SMA at 133 mM did not show any light absorption. We can then assume that there should be no inner filter effect in fluorescence measurements. Three experiments were recorded for each sample (standard deviation <0.03). The LifeSpec II luminescence spectrometer (Edinburgh Instruments), equipped with a 295 nm pulsed light-emitting diode, was used for time-correlated single-photon counting measurements of BSA fluorescence lifetimes at the emission wavelength 340 nm. Data analysis was performed using the FAST software package from

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45

Table 1 Specifications of chemicals used in the present study. Compounds

Source

CAS no.

Purification method

Mass fraction purity

Bovine serum albumin (BSA) Disuccinylmaslinic acid (SMA) Sodium chloride (NaCl)

Sigma–Aldrich Semisynthesized in the laboratorya Fluka

[9048-46-8] ... [7647-14-5]

None Flash-column chromatography None

98% 90% 99.5%

a

Semisynthesis procedure described in Section 2.2.

Edinburgh Instruments. Biexponential functions were used to fit the fluorescence decays, and the fit goodness was evaluated by x2 values and visual inspection of residuals. Average fluorescence lifetimes (t ) were calculated from the two-component contributions using the following equation [15]: X Ai t 2i i t¼X

Ai t i

(1)

i

where Ai is a pre-exponential factor for the component i with a lifetime t i. The calculated average fluorescence lifetime is the result of three experiments (standard deviation <0.05). 1.4. Electrophoretic mobility measurements The electrophoretic mobility (me) was determined by laser Doppler electrophoresis and the measurements were performed on a Zetasizer Nano Series-ZS (Malvern Instrument, Malvern, UK) with dispersion technology software (DTS) at 298.15 K. The electrophoretic mobility was measured as a function of pH while maintaining a constant low ionic strength (2 mM). Each reported value of me is the average of five individual measurements. 2. Results and discussion 2.1. Fluorescence quenching of BSA by SMA The fluorescence spectra of a fixed concentration of BSA (25 mM) in the presence of different concentrations of SMA at pH 7.4, ionic strength 2 mM, and 298.15 K are shown in Fig. 1. This figure shows that BSA has a broad emission band centered at

[(Fig._1)TD$IG]

341 nm after being excited at 295 nm. Moreover, it is clear that the intrinsic fluorescence of BSA is affected by SMA addition. Specifically, the fluorescence intensity progressively declines, accompanied by a blue shift in the emission maximum (see inset in Fig. 1). These data suggest an interaction between SMA and BSA, probably prompting the formation of a non-fluorescent complex. The blue shift detected can be attributed to the formation of a less polar environment around the Trp residues as a result of a more compact conformation of BSA upon substrate interaction. It bears noting that similar qualitative results (data not shown) were found at an ionic strength of 20 and 40 nm. The mechanisms of fluorescence quenching are usually classified into dynamic quenching and static quenching. Dynamic quenching refers to a process where the fluorophore and the quencher come into contact during the lifetime of the excited state. By contrast, the static quenching refers to fluorophore–quencher complex formation. Dynamic and static quenching can be distinguished by their different dependence on temperature and excited-state lifetime. For dynamic quenching, higher temperatures result in faster diffusion and larger amounts of collisional quenching so that the quenching constant increases with temperature. On the other hand, increasing temperature is likely to decrease the stability of complexes, and thus lower the values of the static quenching constant [24]. To clarify the quenching mechanism, the Stern–Volmer equation can be used in the analysis of the quenching data [24]: F0 ¼ 1 þ K SV ½Q ¼ 1 þ K q t 0 ½Q F

(2)

where F0 and F denote the fluorescence intensities in the absence and presence of quencher, respectively; KSV is Stern–Volmer quenching constant; [Q] is the concentration of the quencher; Kq is the biomolecular quenching rate constant; and t 0 is the average lifetime of the molecule without the quencher. From the Stern– Volmer plots of the quenching of BSA fluorescence by SMA at different temperatures, pH 7.4, and ionic strength 2 mM (data not shown), we have calculated the KSV values 2409  110 M1, 1996  85 M1 and 1685  53 M1 for 298.15 K, 305.15 K, and 310.15 K, respectively. The average lifetime of BSA at 298.15 K was measured by time-resolved fluorescence (see below). The calculated value of t 0 was 5.63 ns. According to, Kq = KSV/t 0, the quenching rate constant Kq is 4.3  1011 l mol1 s1. As a rule, for dynamic quenching, the maximum collision quenching constant of various quenchers is 2.0  1010 l mol1 s1 [24]. Clearly, the rate constant of the protein quenching procedure initiated by SMA is higher than this value, indicating that the quenching process is started by the formation of a complex. Moreover, the decrease of the Stern–Volmer constant as temperature rises corroborates this point. 2.2. Ionic strength influence on the binding between SMA and BSA

Fig. 1. Fluorescence emission spectra of BSA (25 mM), after excitation at 295 nm, with different concentrations of SMA at pH 7.4, ionic strength 2 mM, and 298.15 K. Inset shows the variation of emission maxima with SMA concentration. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)

Disuccinylmaslinic acid molecule presents three carboxylic groups which will be deprotonated above certain pH values. We studied the influence of salt concentration on the binding process to assess the role of electrostatic interactions due to these charges.

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Three sets of experiments were conducted with varying NaCl concentrations (2, 20, and 40 mM) at pH 7.4. The obtained quenching data were used to determine the binding constants, Ka, between SMA and BSA from the Lineweaver–Burk equation [18,22,25]: 1

DF

¼

1 1 1 þ F 0 F 0 K a ½Q

(3)

where F0 is the fluorescence intensity in the absence of quencher, F is the fluorescence intensity in the presence of a molar concentration of quencher [Q], and DF = F0  F. Thus, a linear plot of 1/DF vs. 1/[Q], in accordance with the above equation, provides F0 from the intercept on the ordinate and the slope gives the estimate for the binding constant. Fig. 2 shows our data for 2 mM and pH 7.4 plotted according to Eq. (3), which are found to be linear (r2 > 0.998), indicating that the assumptions underlying the derivation of Eq. (3) are satisfactory. The calculated values of the binding constants, Ka, in 2, 20, and 40 mM of NaCl as a function of temperature are listed in Table 2. It was observed that the binding constant between SMA and BSA was moderate, and for a given temperature the strength of SMA coupling to BSA was reduced with electrolyte concentration. Binding between the drug and protein may arise from different forces, namely hydrogen bonds, electrostatic, van der Waals, and hydrophobic interactions [26]. These factors contribute to the affinity constant and energetic of the binding process. The influence of electrolyte on this constant is often used to assess the role of electrostatic interaction on the association process [27], as an increase in electrolyte concentration reduces the contribution of this interaction to the binding of Gibbs energy and thus weakens binding. Our results suggest that electrostatic interactions contribute to SMA–BSA binding, since they are screened by the presence of NaCl in the medium. SMA has three carboxylic groups which, probably, at pH 7.4 are deprotonated (pKs of succinic and maslinic acid are 4.2, 5.7 and 4.4, respectively) [28,29]. This negatively charged SMA molecule may interact with the positive residues of the protein, and the interaction be attenuated by the presence of the NaCl in solution. It is noteworthy that when NaCl concentration rose from 2 mM to 20 mM, Ka dramatically fell (81% for all temperatures studied). It is likely that at low NaCl

concentration the electrostatic interactions were the major contributors to the binding process. On the other hand, Ka was reduced to 31% at 298.15 K when NaCl concentration rose from 20 mM to 40 mM. This suggests that at 20 mM, NaCl electrostatic interactions were not a driving force in the binding, and other interactions predominated. As reflected in Table 1 2, the decreasing trend of Ka with temperature was stronger as the salinity increased. Again, these data suggest that the nature of molecular forces controlling the SMA–BSA binding differ at low and high electrolyte concentrations. The ion linkage numbers were determined from the slope of the plots of log Ka vs. log [NaCl] at each temperature (data not shown) [30]. The slopes were calculated from the linear regressions of these plots, being: 0.68  0.03, 0.69  0.01, and 0.74  0.02 for 298.15 K, 305.15 K and 310.15 K, respectively. The negative sign indicates that the counterions are released upon formation of the SMA–BSA complex [31]. These data suggest that the exchange of counterions between the protein and solvent is favored by temperature. It is noteworthy that the calculated slope values for our system lie within the range already reported for the binding of different ligands to albumin [32]. The sign and magnitude of the changes in enthalpy (DH0) and entropy (DS0) components of Gibbs energy can be informative regarding the interactions acting in drug–protein binding. Ross and Subramanian have proposed a possible identification of the forces that are key in ligand binding from the analysis of thermodynamic parameters [33]. According to this approach, negative DH0 and positive DS0 indicate that electrostatic interaction plays a major role in the binding process; positive DH0 and DS0 would be considered to be evidence for hydrophobic interaction, whereas negative values of both thermodynamic parameters arise from van der Waals forces and hydrogen bonds. To elucidate the interaction types controlling the binding between SMA and BSA under the different experimental conditions studied, we calculated the thermodynamic parameters, i.e. Gibbs energy changes, DG0, enthalpy changes, DH0, and entropy changes, DS0, from the following equations:

DG0 ¼ RTlnK a 2  0 . 3 d DG T 5 DH0 ¼ T 2 4 dT

[(Fig._2)TD$IG]

T DS0 ¼ DH0  DG0

Fig. 2. Quenching data plotted according to Eq. (3) for the binding of SMA to BSA at different temperatures, pH 7.4 and ionic strength 2 mM.

(4)

(5)

(6)

Thus, we found the DG0 values by substituting the values of Ka in Eq. (4), and then, from the slopes of the plots of DG0/T vs. T (see Fig. 3), we estimated DH0 by Eq. (5), and finally, the corresponding entropic contributions were calculated from Eq. (6). The thermodynamic parameters calculated this way are listed in Table 2 and plotted in Fig. 4 for a better visualization. As can be seen, for a given temperature the presence of NaCl in the medium made the association process less favorable (i.e. the magnitude of DG0 decreased). On the other hand, at low ionic strength, temperature favored (although slightly) the binding of SMA to BSA, whereas at 20 mM and 40 mM the spontaneity diminished with temperature. Under all the experimental conditions studied the binding process proved exothermic (negative value of DH0) and for all NaCl concentrations the heat released augmented with temperature. Enthalpy change provided a more favorable contribution to binding as the electrolyte concentration increased. On the contrary, at greater NaCl concentrations, the entropic term became

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[(Fig._3)TD$IG]

Fig. 3. Plots of DG0/T vs. T at pH 7.4 and different ionic strengths.

progressively less favorable, although the magnitude of the enthalpic term remained large enough to overcome this unfavorable entropy contribution and keep the binding process spontaneous. At 2 mM NaCl we found that DS0 > 0, confirming the dominance of electrostatic interactions in the binding of SMA to BSA. This gain in entropy was the consequence of the disruption of the hydrogenbonding network between water molecules and the protein by the SMA molecules. The release of water molecules that hydrate drug and protein into bulk solvent was accompanied by a favorable entropy change. Nevertheless, at 20 mM and 40 mM, DS0 < 0, suggesting that hydrogen bonds and van der Waals forces control the association process. As stated above, the electrostatic forces gradually weakened as the NaCl concentration rose, with other interactions as driving forces emerging in the binding of SMA to BSA. Information concerning the heat-capacity change (DCp) of the association process can be gained from the temperature-dependent enthalpy change [32,34]. It is worth noting the good linear relationship between DH0 and temperature for all NaCl concentrations, as shown in Fig. 5 (r2 > 0.9998). The slope of the plot (dDH0/dT) is a measure of the heat-capacity change between the initial and final states of the system (DCp). The linearity of the plot suggests that DCp is independent of temperature. The values evaluated for DCp from linear-regression analysis are listed Table 2.

47

Heat-capacity values provide a link between structural and energetic data. A solvent reorganization at the protein surface, upon drug–protein complex formation, is generally considered to be the most significant contributor to DCp [35]. In particular, negative DCp values indicate that the protein surface is removed from contact with solvent when it is associated with the drug to form a complex [36,37]. Inspection of the values of DCp reveals that as the NaCl concentration increases the exposure of protein hydrophobic patches to water decreases, suggesting a loss of solvating water structure in the association process. Furthermore, electrostatic forces provide a positive term to DCp and this may cause the rise in DCp (in absolute value) with electrolyte concentration [38]. 2.3 pH influence on the binding between SMA and BSA To gain insight into the correlation between chemical characteristics and binding affinity of SMA to BSA, we performed fluorescence titrations at five different pHs at 2 mM of ionic strength (viz., 2.8, 4.2, 6.5, 7.4, and 9.1). Under these experimental conditions, both protein and drug charges will depend on pH values. The temperature of the solutions varied from 298.15 K to 310.15 K. Table 3 gives the values of Ka obtained from the respective quenching fluorescence curves. Thermodynamic parameters calculated from Eqs. (4)–(6) (DG0, DH0 and DS0) are also listed in this table. As can be seen, SMA exhibited the strongest affinity towards BSA at pH 7.4. At lower pH values, the interaction diminishes markedly, being similar at pH 2.8 and 4.2. This result suggests that the ionized SMA form is more favorable to interact with the protein. Silva et al. indicated that at pH 7.4, the cavity in the BSA molecule where the Trp 214 residue is located contains numerous positively charged arginine and lysine residues, thus creating a favorable environment for the negative SMA molecule to bind [39]. The number of these positive residues is reduced at pH 9.1 where BSA is in an extremely negative charged form. Consequently, Ka dramatically decreases at this pH. In acidic medium, pH 2.8 and 4.2, the SMA molecule is uncharged. Therefore the possible electrostatic attractions disappear, in turn giving rise to a lower constant binding. These explanations are corroborated from the analysis of thermodynamic parameters at each pH. From the sign of DH0 and DS0 (see Table 3), it is deduced that at pH values lower than the isoelectric point of BSA (i.e.p. 4.9) the dominant acting forces between SMA and BSA are hydrogen bonds and van der Waals interactions. Otherwise, at pH values higher than i.e.p. the electrostatic forces control the attachment of the drug to the protein. Fig. 6 illustrates the values of the thermodynamic functions for the binding of SMA to BSA as a function of pH. The negative sign of DG0 confirms the statement that the association processes were all spontaneous. The magnitude of DG0 at pH above the i.e.p. was

Table 2 Binding constants and thermodynamic parameters for the interaction between SMA and BSA at pH 7.4 as a function of the ionic strength and temperature at pressure of 0.1 MPaa . T /K

Ka  104/M1

DG0(a)/(kJ mol1)

DH0(b)/ (kJ mol1)

2

298.15 305.15 310.15

3.72  0.51 2.97  0.34 2.57  0.21

26.07 26.11 26.17

22.81 23.90 24.69

3.26 2.21 1.48

0.156

20

298.15 305.15 310.15

0.72  0.09 0.59  0.07 0.49  0.04

22.02 22.04 21.89

23.98 25.12 25.95

1.96 3.08 4.06

0.164

40

298.15 305.15 310.15

0.50  0.07 0.38  0.05 0.28  0.02

21.09 20.87 20.45

34.63 36.28 37.28

13.53 15.40 17.02

0.237

Ionic strength/mM

TDS0(c)/(kJ mol1)

DCP (d)/(kJ mol1 K1)

a The standard uncertainty u is u(T) =  0.1 K; the combined expanded uncertainties u are u(Ka) =  . . . ; u(DG0) =  0.1 kJ/mol; u(DH0) =  1.0 kJ/mol; u(TDS0) =  1.0 kJ/ mol; and u(DCP) =  0.001 kJ mol1 K1 (0.95 level of confidence).

48

[(Fig._4)TD$IG]

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Fig. 4. Effect of the ionic strength on standard Gibbs energy, DG0, enthalpy change, DH0, and entropy change, DS0, for the binding of SMA to BSA at pH 7.4 and different temperatures.

[(Fig._5)TD$IG]

Fig. 5. Enthalpy change as a function of temperature for the binding of SMA to BSA at pH 7.4 and different ionic strengths.

higher than that for pH values below the i.e.p. In other words, when electrostatic forces were the dominant interaction, the attachment of SMA to BSA was more favorable. Notably, when temperature was raised the spontaneity of binding decreased if hydrogen bonds and van der Waals forces dominated the process. By contrast, DG0 became more negative with temperature for pH values above the i.e.p. When hydrogen bonds and van der Waals forces played a major role in the formation of drug–protein complexes (pH 2.8 and 4.2), the maximum (absolute) DH0 and DS0 values were reached. The negative DH0 indicates that the binding process is accompanied by heat release in all the experimental conditions studied. Electrostatic interactions cause an exothermic effect [40]. On the other hand, the expulsion of water molecules from protein to bulk solution when hydrogen bonds between SMA and BSA are formed is also exothermic [41]. This released water makes additional hydrogen bonds upon returning to the bulk solvent, thus emitting heat. As can be inferred from Table 3 and Fig. 6, the magnitude of enthalpy change was considerably higher when the binding process was mainly controlled by hydrogen bonds (pH 2.8 and 4.2) than when the SMA–BSA association was dominated by electrostatic forces (pH 6.5, 7.4, and 9.1). Electrostatic interactions are expected to contribute little to the binding enthalpy in water solution [42,43]. For all the pH values studied, the value of DH0 (in absolute value) increased when temperature rose. In terms of water structure, a negative DS0 value is often ascribed to the formation of hydrogen bonds and van der Waals forces. Electrostatic interactions, however, are characterized by a

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49

Table 3 Binding constants and thermodynamic parameters for the interaction between SMA and BSA at ionic strength 2 mM as a function of pH and temperature at pressure of 0.1 MPaa . pH

T/K

Ka  104/M1

DG0(a)/(kJ mol1)

DH0(b)/ (kJ mol1)

TDS0(c)/(kJ mol1)

DCP (d)/(kJ mol1 K1)

2.8

298.15 305.15 310.15

0.21  0.03 0.14  0.01 0.10  0.01

18.93 18.30 17.75

46.15 48.35 49.94

27.22 30.05 32.19

0.316

4.2

298.15 305.15 310.15

0.22  0.03 0.15  0.02 0.11  0.01

19.05 18.54 17.61

51.95 54.42 56.22

32.90 35.88 38.61

0.356

6.5

298.15 305.15 310.15

0.88  0.09 0.80  0.07 0.72  0.03

22.49 22.79 22.89

11.90 12.47 12.88

10.58 10.32 10.00

0.082

7.4

298.15 305.15 310.15

3.72  0.51 2.97  0.34 2.57  0.21

26.07 26.11 26.17

22.81 23.90 24.69

3.26 2.21 1.48

0.156

9.1

298.15 305.15 310.15

1.48  0.18 1.34  0.12 1.19  0.12

23.79 24.10 24.19

13.32 13.95 14.42

10.47 10.15 9.77n

0.092

a The standard uncertainty u is u(T) =  0.1 K; the combined expanded uncertainties u are u(Ka) =  . . . ; uDG0) =  0.1 kJ/mol; u(DH0) =  1.0 kJ/mol; u(TDS0) =  1.0 kJ/ mol; and u(DCP) =  0.001 kJ mol1 K1 (0.95 level of confidence).

positive value of DS0. If these interactions are attenuated or eliminated, the final value of DS0 will be more negative. Note that the magnitude of DS0 decreases with increasing temperature for binding processes dominated by electrostatic forces. Conversely, the opposite trend is observed when the association processes are

[(Fig._6)TD$IG]

controlled by hydrogen bonds. For these samples, larger negative values of DCp have been deduced. Alterations of protein conformation with pH could also affect the binding of SMA to the protein. BSA is known to undergo several pH-dependent transitions. The normal (N) form is predominant

Fig. 6. Effect of the pH on standard Gibbs energy, DG0, enthalpy change, DH0, and entropy change, DS0, for the binding of SMA to BSA at ionic strength of 2 mM and different temperatures.

50

[(Fig._7)TD$IG]

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Fig. 7. (a) Fluorescence decay curves (lexc = 295 nm, lem = 341 nm) of BSA at different pHs. (b) Fluorescence intensity maxima and average lifetime values of BSA, t , as a function of pH.

from pH 4.5 to 7.0. The conformation of BSA undergoes a transformation from a normal (N) to a fast-moving state (F) when the solution pH is lowered from 4.5 to 4. At pH < 4, the expanded form appears (E). On the basic side of pH, another conformational change occurs between pH 8.0 and 9.0 (the so-called N–B transition) [44]. The shape of BSA molecules becomes globular in the N-form, its conformation being partly opened in the F-form. In the B-form the albumin loses some of its rigidity. The observed reduction in the binding constants from pH 7.4 to pH 2.8 may result from these alterations of the protein structure induced by the pH change. To illustrate these changes in the BSA conformation, steady and time-resolved fluorescence studies of BSA solution at different pHs were performed. The results are presented in Fig. 7, where maximum fluorescence intensity (Fmax) and mean fluorescence lifetime (t ) are plotted as a function of pH. It can be inferred that Fmax and t values at pH 6.5 are greater than those at other pH values. The change in both parameters reflects that pH variation leads to conformational transitions of the protein, and thereby the attachment capacity of SMA to BSA may be affected. The lowest values of Fmax and t were measured at extreme pH values (2.8 and 9) where the albumin macromolecule is characterized by a loosening structure. It has been reported that under these conditions the protein presents a greater surface area as compared to the N-form [44]. Fig. 8 depicts DH0 plotted against DS0 for all experimental conditions examined. As can be seen in the data fit with a linear

[(Fig._8)TD$IG]

regression (r2 > 0.998). This linearity, known in literature as enthalpy–entropy compensation, is an important characteristic of protein-binding interactions [45]. This compensation has proven to be rather common for processes occurring in aqueous media and is connected with water reorganization accompanying protein–ligand interactions [46]. It is generally agreed that any changes in the hydrogen-bonding arrangement of water molecules will produce nearly, or exactly, compensating changes in both enthalpy and entropy [47]. Such changes should therefore not prompt a large free-energy change. The phenomena observed in Fig. 8a are striking that is, all the processes where hydrogen bonds are the major binding forces fall in the same compensation line. Breiten et al. have recently suggested that changes in the structure of hydrogen-bonded water networks, which result after ligand bindings, determine the values DH0 and DS0, and then the enthalpy–entropy compensation [48]. Probably, this water reorganization is similar when SMA linking to BSA occurs predominantly by hydrogen bonds (pH 2.8, pH 4.2, and pH 7.4 at 20 mM and 40 mM NaCl). On the other hand, inspection of DCp values reported in Tables 2 and 3, reveals that the lower values have been found for experimental conditions where electrostatic interactions control the binding process. It is likely that in these association processes different types of minor water reorganization take place. The enthalpy and entropy changes are usually related by the relationship [49,50]:

Fig. 8. Enthalpy–entropy compensation plots for the binding of SMA to BSA under different conditions: (a) binding processes controlled mainly by van der Waals and hydrogen bonds interactions, and (b) binding processes controlled mainly by electrostatic interactions.

J.A. Molina-Bolívar et al. / Fluid Phase Equilibria 400 (2015) 43–52 0



DH ¼ DH þ T C DS

0

(7)

where TC is the compensation temperature, that is explained as a feature of protein–water interaction. At this temperature, the enthalpy and entropy changes are cancelled out completely. The values of TC determined from the slope of the plots in Fig. 8b, expressed in Kelvin, are 303  6, 304  3, and 305  5 for pH 6.5, 7.4, and 9.1, respectively. The calculated value of TC from Fig. 8a was 268  3. This value, attributed to the complex formation under conditions where hydrogen bonds are the dominant forces, is lower than those obtained for situations where electrostatic interactions control the binding process. The compensation temperature corresponds to the particular temperature at which the process is purely enthalpy-driven (DG0 = DH*). Higher values of TC indicate that more energy is required to induce the release of a certain number of water molecules. It is noteworthy that binding processes with lower magnitude of DCp correspond with higher TC. Our TC values are found to lie well within 250–325 K (the reported range where water reorganization is associated with protein unfolding, protein–ligand interaction, etc.) [51]. The intersection of the compensation plot, DH*, provides the enthalpy effect under the condition of zero entropy change, and therefore characterizes the drug–protein interactions. The evaluated values of DH* from linear regression are 22.7  0.6 kJ/mol, 26.1  0.1 kJ/mol, and 24.1  0.7 kJ/mol for pH 6.5, 7.4, and 9.1, respectively, indicating that the same influence of pH on Ka is observed in DH*. Indeed, the higher affinity of SMA to BSA and the higher DH*, in absolute value, correspond to pH 7.4. 2.4. Detection of complex formation by electrophoretic mobility The surface charge of protein molecules is affected by pH. Depending on this value, amino and carboxylic groups are ionized, resulting in a net charge that can be revealed by electrophoretic mobility measurements. Another factor that can modify the net charge of a protein is the accumulation of charged ligands on the protein surface [52]. In order to confirm the binding of SMA to BSA, the electrophoretic mobility of SMA–BSA complexes at pH 9.1 and pH 2.8 has been measured (see Fig. 9). As expected, at a pH value higher than the isoelectric point of BSA (i.e.p. is 4.9) the net charge

[(Fig._9)TD$IG]

51

of the complex is negative, whereas at a pH lower than the i.e.p. the electrophoretic mobility is positive. At pH 9.1, pure BSA molecules, in the absence of added SMA, showed a me value of 0.98 m2 s2 V1. As the drug concentration increases, the magnitude of electrophoretic mobility of the complex also increases up to 1.78 m2 s2 V1 for 133 mM of SMA. This result supports the idea of a complex formation: in this alkaline medium the drug is completely deprotonated and the binding of SMA to BSA provides an increase of the negative charge of BSA. It should be borne in mind that each SMA molecule adsorbed onto the protein contributes three negative charges. It is noteworthy that the curve displays a plateau at high SMA concentrations, suggesting saturation in the drug binding to BSA. With regard to pH 2.8, the electrophoretic mobility is constant for the entire SMA concentration range studied. In this case, the binding of SMA molecules does not alter the electrical state of the protein, since SMA is protonated, and then uncharged, in this acid medium. 3. Conclusions The optimization of drug design requires the knowledge of the influence of ligand structure on the affinity to proteins. For this, a thermodynamic analysis of binding processes was performed. In this context, we used fluorescence spectroscopic methods to study the interaction of the drug disuccinylmaslinic acid (SMA) and bovine serum albumin (BSA) at different pH values and NaCl concentrations. Albumin fluorescence exhibits a reduction in the intensity upon the binding of SMA. The emission maximum shows a slight blue shift, indicating that the hydrophobicity in the vicinity of the fluorophore residues increases as a consequence of drug attachment. In order to assess the influence of electrostatic interaction on the formation of SMA–BSA complexes at pH 7.4, binding constants have been determined as a function of NaCl concentration. The affinity decreases with increasing ionic strength, the association process being less favorable. This reduction is due to the screening of electrostatic interaction. Regarding the pH influence on the binding process, it has been observed that at pH values lower than the isoelectric point of albumin (i.e.p. 4.9), the dominant acting forces were hydrogen bonds and van der Waals interactions (pH 2.8 and 4.2). On the other hand, at pH values higher than i.e.p. the electrostatic force dominated the process. In these cases, temperature favored the SMA–BSA complex formation, as opposed to the behavior observed at low pH values. Under all experimental conditions examined the process was accompanied by heat release. Heat-capacity change (DCp), estimated from the enthalpy temperature dependence, showed higher values when hydrogen bonds and van der Waals interactions were the driving force in the SMA–BSA association. Our study found that an enthalpy–entropy compensation relationship holds for this process. It is remarkable that binding processes controlled mainly by hydrogen bonds and van der Waals interactions (pH 2.8, 4.2, and 7.4 at high NaCl concentrations) fall in the same compensation line. The observation of this entropy-enthalpy compensation suggests that water reorganization plays an important role in the binding of SMA to BSA. Changes in electrophoretic mobility of albumin as a function of drug concentration corroborate the complex formation. Acknowledgements We thank David Nesbitt for reviewing the English in the manuscript. References

Fig. 9. Electrophoretic mobility of the SMA–BSA complexes as a function of the SMA concentration at pH 2.8 and 9.1.

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