Assessment of the Europium(III) Binding Sites on Albumin Using

May 22, 2014 - Intrinsic fluorescence quenching of bovine serum albumin (BSA) and europium(III) luminescence in BSA complexes were investigated. The n...
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Assessment of the Europium(III) Binding Sites on Albumin Using Fluorescence Spectroscopy Tatiana N. Tikhonova,† Evgeny A. Shirshin,*,‡ Gleb S. Budylin,‡ Victor V. Fadeev,‡ and Galina P. Petrova‡ †

International Laser Center and ‡Department of Physics, M.V. Lomonosov Moscow State University, Moscow 119991, Russia S Supporting Information *

ABSTRACT: Intrinsic fluorescence quenching of bovine serum albumin (BSA) and europium(III) luminescence in BSA complexes were investigated. The number of BSA binding sites (n) and equilibrium constant (Keq) values were determined from both measurements provided qualitatively different results. While the modified Stern−Volmer relation for BSA fluorescence quenching gave n = 1 at pH 4.5 and pH 6, two sets of binding sites were determined from Eu3+ luminescence with n1 = 2, n2 = 4 at pH 6 and n1 = 1, n2 = 2 at pH 4.5. The model explaining the discrepancy between the results obtained by these fluorescent approaches was suggested, and the limitations in application of the “log−log” Stern− Volmer plots in analysis of binding processes were discussed.

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this case, a single binding site is usually indicated.5,6,13,18−22 Moreover, the analysis of about 65 works, in which albumin’s affinity for a variety of substances of different origin (drugs, toxins, quantum dots, etc.) is studied and where this process is characterized with the use of FQT and the modified Stern− Volmer equation, showed that in all of these works the number of binding sites is n = 1 despite the dramatic differences between the systems under investigation. The same fact was also denoted in the study of Lissi and Abuin.23 Here, we will try to explain the observed discordance. First, we investigated experimentally the interaction between BSA and europium(III) ions in aqueous solution, using both intrinsic protein fluorescence quenching and europium(III) luminescence, to determine n and K e q values. Eu 3+ luminescence sensitivity to its coordination allowed the number of bound ions to be obtained and its binding to BSA to be assessed quantitatively, including the use of Scatchard plots.24 The results of this investigation were compared to the data provided by FQT. Second, we performed the numerical modeling of the binding process based on the model of independent sites, used in the derivation of the modified Stern−Volmer equation, and analyzed the reasons of inadequate n values determination.

INTRODUCTION Interaction between metal ions and proteins has been studied over decades.1−3 The significance of this process is caused by its importance in human metabolism4 and by the possible toxicity of metal ions that can initiate protein conformational changes, denaturation,5,6 and aggregation.7−9 Fluorescence spectroscopy, the method that provides information about the changes in local environment of fluorophores, is extensively used to study metal binding by proteins.10−12 The basic characteristics of the binding processthe number n of binding sites and the equilibrium constants Keqcan be obtained using the fluorescence quenching technique (FQT),13 e.g., by the analysis of the modified (double logarithmic or “log−log”) Stern− Volmer plots. The analysis of literature devoted to interaction of proteins (by the example of albumin, the most abundant protein in blood plasma) with different metal ions using various methods strongly suggests that the number of binding sites n exceeds unity. For instance, Masuoka et al.14 stated that “the literature reveals a chronological trend toward a lower number of binding sites n due to a shift in modeling from many, simple sites (e.g. one metal atom per surface residue) to fewer, more complex sites”. Without focusing on the nature of metal binding sites in albumin, it can be summarized that the well-accepted concept postulates the existence of one15 or two16 high-affinity specific sites that attract the primary attention of researchers due to their physiological relevance, and a group of nonspecific sites that is usually ignored. Another situation is observed when the fluorescence quenching technique is used for the quantitative assessment of metal binding by albumin. Since the Stern−Volmer equation17 does not provide the number of binding sites, its modified, double logarithmic form13 is used for this purpose. In © 2014 American Chemical Society

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MATERIALS AND METHODS Sample Preparation. Bovine serum albumin (BSA) and europium nitrate (Eu(NO3)3·5H2O) were purchased from Sigma (Germany). Other chemicals were reagent grade. BSA and europium nitrate stock solutions of 0.1 and 10 mM, Received: February 5, 2014 Revised: May 21, 2014 Published: May 22, 2014 6626

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respectively, were prepared in bidistilled water. All the experiments were performed at ambient temperature (25 ± 2 °C). Two series of experiments were performed at pH 6 and pH 4.5. The pH values were set with hydrogen chloride. The ionic strength of all the samples was fixed to 0.1 M by sodium chloride. BSA concentration was determined spectrophotometrically using an extinction coefficient of 43 824 M−1cm−1 at 280 nm.25 Experimental Conditions. Here, we did not use buffer solutions to fix the pH value because of the possible complex formation between Eu3+ and buffer components; e.g., it was shown in the article of Pfefferle and Bunzli26 that the equilibrium constant for Tris−HCl buffer and Eu3+ complexation was log Keq = 2.4. It is noteworthy that the same situation holds for other heavy metal ions (e.g., Pb2+, Zn2+, etc.27−29), though in this case it is more difficult to control complexation with buffer solutions because these ions do not obtain luminescence and this effect can be erroneously omitted. To avoid the formation of Eu3+ coordination compounds with buffer components, the experiments were carried out in aqueous solution and the pH value was set with HCl. Upon addition of the maximum europium(III) concentration used in the experiments ([Eu3+] = 3.5 × 10−4 M) to BSA, we observed a minor decrease of the pH (from 6.3 down to 6.1). Interaction processes in the model system of protein and metal ions are usually studied at a pH close to normal (7.4). At this pH value, Eu3+ hydration occurs,26 thus resulting in the appearance of competing complexation processes (Eu 3+ complex formation with BSA and OH− ions). This makes the system more complex and can lead to errors in K eq determination. Thus, the upper limit for pH values in our experiments was equal to 6, where the BSA conformation was the same as at pH 7.4 and no Eu3+ hydroxides were presented in the solution. Eu3+−BSA binding was studied at two pH values, where BSA has negative (pH 6) and positive (pH 4.5) net charge.30 Fluorescence and Absorption Measurements. Fluorescence spectra were recorded on a FluoroMax-4 spectrofluorometer (Horiba Jobin-Yvon, Japan-France) with 150 W xenon lamp. Measurements were carried out in a 3.5 mL quartz cuvette at constant stirring of the sample. Eu3+ luminescence spectra were obtained at 394 nm excitation in the 500−720 nm range; excitation and emission slit widths were 5 and 10 nm, respectively. BSA fluorescence emission spectra were obtained at 295 nm excitation in the 310−500 nm range; excitation and emission slit widths were 1 and 5 nm, respectively. At this excitation wavelength, BSA fluorescence is due to two tryptophan residues.20 Fluorescence measurements included titration of BSA aqueous solution with Eu3+ aliquots and vice versa. The pH value of all solutions was measured using an Aquilon pH meter (Aquilon, Russia). The optical density of the solutions was obtained using a Lambda-25 spectrophotometer (PerkinElmer, Germany) in the 200−600 nm range.

Figure 1. Dependency of optical density for the BSA−Eu3+ system on the Eu3+ concentration measured at 500 nm. [BSA] = 4.3 × 10−5 M.

Hence, we performed all the fluorescence measurements in the [Eu3+] concentration range where aggregation can be neglected, and the optical density at 295 nm is constant. Intrinsic BSA Fluorescence Quenching. Intrinsic BSA fluorescence quenching upon titration with Eu3+ was studied to assess the binding process quantitatively. The fluorescence quenching technique is widely used to obtain protein binding affinities for different substances.5,6,13,18−22 BSA fluorescence emission at 295 nm excitation is due to the presence of two tryptophan residues; the first one is located near the surface (Trp-134) and the second (Trp-212), in the internal hydrophobic region.31 On Eu3+ addition, intrinsic BSA fluorescence decrease is observed (see Figure 2A). This effect can be caused by direct interaction of Eu3+ ions with BSA fluorophoresTrp-212 and/ or Trp-134 residues.10 Another explanation could include metal-induced conformational changes of BSA, leading to the changes in the environment of tryptophan residues.6,11 We followed the standard procedure used for quantitative assessment of binding processes between protein and metal ions using the FQT. This approach implies the use of the Stern−Volmer equation assuming fluorescence quenching to be static, i.e., considering the fluorescence quantum yield of Eu3+− BSA complexes to be zero:17 F0/F = 1 + KSV[Q]

(1)

where F0 and F stand for the fluorescence intensities in the absence and in the presence of quencher, [Q] is the concentration of quencher (in our case, [Eu3+]), and KSV is the Stern−Volmer quenching constant. The number of binding sites n and equilibrium constant Keq can be determined using a modified “log−log” Stern−Volmer equation:13 F0 − F = log Keq + n log[Q] (2) F Equation 2 is also obtained under the assumption of static quenching of BSA upon ligand binding. The modified Stern−Volmer plots are presented in Figure 2B for the system of BSA and Eu3+ at two pH values (6 and 4.5). Equation 2 gives for the “log−log” Stern−Volmer plots the number of binding sites n = 1 for both pH values, while the quenching constants are log Keq = 3.1 and log Keq = 2.9 at pH 4.5 and pH 6, respectively. Eu3+ Fluorescence Studies. Another approach used in this work for the determination of n and Keq was based on Eu3+ luminescence measurement in BSA−Eu3+ complexes. log

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RESULTS Optical Density Measurements. The optical density spectra of the BSA−Eu3+ solutions were measured. It was observed that at a certain metal to protein ratio the increase in optical density measured at wavelength λ = 500 nm, that can be attributed to protein aggregation, took place (see Figure 1). 6627

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Figure 2. (A) Emission spectra of BSA ([BSA]T = 4.3 × 10−5 M) at excitation wavelength λex = 295 nm at different Eu3+ concentrations. (B) The “log−log” Stern−Volmer plots for BSA fluorescence quenching at different pH’s.

Figure 3. (A) Eu3+ fluorescence spectra in aqueous solution (dashed line), [Eu3+]T = 10−4 M and in the presence of BSA (solid line), [BSA]T = 4 × 10−5 M, [Eu3+]T = 10−4 M. (B) Dependence of the ratio of the 594 nm to the 616 nm peaks in the Eu3+ spectrum on BSA concentration for pH 6 (black squares) and pH 4.5 (red triangles), [Eu3+]T = 10−4 M.

binds four Eu3+ ions (n = 4), while at pH 4.5 the number of bound ions decreases to two per protein molecule (n = 2). Representation of Eu3+ Luminescence Data in the Form of Scatchard Plots. Investigation of ligand binding by proteins can also be performed on the basis of the Scatchard model.36 This approach allows the coexistence of sites with different binding affinities within a single molecule. According to the Scatchard model, the average number of bound Eu3+ ions per BSA molecule ν can be expressed as

Lanthanide ions are extensively used as luminescent probes for the investigation of different biological systems. 32 Luminescence of Eu3+ is predominantly a result of transitions from its 5D0 to all of the lower lying 7FJ electronic states.33 The ratio of peak intensities at 594 and 616 nm in Eu 3+ luminescence spectrum is sensitive to its coordination and can be used as an indicator of the binding process.34 These peaks correspond to magnetic dipole and induced electric dipole transitions, respectively. To study the binding process, Eu3+ aqueous solution was titrated with BSA. As shown in Figure 3A, alteration of Eu3+ spectra occurs in the presence of the protein. In aqueous solution, Eu3+ is coordinated with nine water molecules32 and the ratio of peak intensities at 594−616 nm is I594/I616 = 2.4. Then, on the increase of BSA concentration in solution, the intensity of the peak at 594 nm, corresponding to the magnetic dipole 5D0−7F1 transition, remains almost constant, while the intensity of the peak at 616 nm, corresponding to the 5D0−7F2 “hypersensitive” transition,35 increases. This fact indicates inner-sphere complex formation between Eu3+ and BSA. The dependence of the ratio of peak intensities at 594−616 nm on BSA concentration for two pH values is shown in Figure 3B. The BSA concentration at which the I594/I616 value reaches a plateau corresponds to the situation when all Eu3+ ions are bound by protein molecules, and hence, no further variation of the luminescence spectrum is observed. As can be seen in Figure 3B, for the curve obtained at pH 6, the ratio I594/I616 becomes constant at [Eu3+]/[BSA] = 4, and at pH 4.5, the I594/I616([BSA]) curve reaches a plateau at [Eu3+]/[BSA] = 2. This means that at pH 6 each BSA molecule

N

ν=

∑ i[Eu iBSA] cB = i = [BSA]T [BSA]T

N

∑ i

i niKeq cF i 1 + Keq cF

(3)

where N is the number of binding site types, [Eui BSA] is the concentration of BSA molecules with i bound ions, ni is the number of binding sites of ith type per single BSA molecule, Kieq is the equilibrium constant for the site of ith type, [BSA]T is the total concentration of protein, cF is the concentration of free europium ions in solution, and cB is the concentration of bound europium ions in solution. If the number of free and bound ligands is known, it is possible to represent data in the form of a Scatchard plot in which the ordinate shows the moles of ligand bound per total moles of protein divided by the concentration of free ligand and the abscissa shows the moles of bound ligand per total moles of protein. Scatchard plots are extensively used for the analysis of calorimetric and dialysis data obtained from protein titration with ligand, e.g., metal ions.14,37 The numbers of free and bound ions were determined via deconvolution of Eu3+ spectra measured at different concentrations of protein using the spectra of free (in aqueous 6628

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Figure 4. Scatchard plots for the BSA−Eu3+ system for pH 6 and pH 4.5.

Table 1. Comparison of Numbers of Binding Sites and Constants of Equilibrium, Obtained by Different Methodsa n method FQT (modifed Stern−Volmer equation) Eu3+ fluorescence (I593/I616 ratio) Scatchard plot a

pH 6 1.2 4.0 1.8 4.4

± ± ± ±

log Keq pH 4.5

0.1 0.3 0.2(1) 0.3(2)

1.3 2.0 1.2 2.4

± ± ± ±

0.1 0.2 0.1(1) 0.2(2)

pH 6

pH 4.5

2.9 ± 0.1

3.1 ± 0.1

5.4 ± 0.3(1) 4.4 ± 0.2(2)

4.4 ± 0.2(1) 3.8 ± 0.1(2)

Upper indexes in the Scatchard plot case denote two different site types.

of n = 4 and log Keq ≈ 4 were obtained. Schomacker et al. studied association constants of lanthanide−albumin complexes by performing equilibrium dialysis experiments and obtained log Keq = 6.41 for Eu3+.39 Ganjali et al. indicated the presence of two binding sites in HSA for Er3+ ion with negative cooperativity and equilibrium constants log K1 = 8.4 and log K2 = 7.9 using a potentiometric membrane microsensor.40 Analogous results were obtained by the same authors using microcalorimetry titrations.41 Finally, interaction of BSA and Eu3+ was studied by microcalorimetry.42 Authors suggested the existence of two classes of binding sites with n1 = 1.7 for highaffinity specific sites and n2 = 6.6 for nonspecific ones. These results are in agreement with the general concept that postulates the coexistence of binding sites with different affinities for the ligand within a single protein molecule. The n values determined from Eu3+ luminescence measurements in this work are in good agreement with the literature data; e.g., the results of Li et al.42 are close to the parameters obtained from the Scatchard plots presented in Figure 4. In contrast, the number of binding sites n obtained from fluorescence quenching curves is equal to unity (Table 1). The latter observation, as stated above, is typical for experiments that deal with fluorescence quenching of protein and the double logarithmic Stern−Volmer equation. Recently, several papers criticizing the use of fluorescence spectroscopy for binding characterization were published. The pitfalls of fluorescence spectroscopy were denoted, including the use of the modified Stern−Volmer equation.43 It was supposed that the modified Stern−Volmer equation was mathematically correct, while the reason for inadequate binding site number determination was supposed to be the discrepancy in full ligand concentration and in its free concentration. The more general criticism of the modified Stern−Volmer equation was provided23the authors, operating with the model of independent binding sites, stated that n in eq 2 did not represent the number of binding sites and can only be used as a characteristic of cooperativity of the binding process. Moreover, the authors23,43 indicated that the use of the model of stepwise

solution) and bound (in excess of BSA) Eu3+ (see Appendix A in the Supporting Information). The results of Eu 3+ luminescence measurements in BSA complexes presented in the form of a Scatchard plot for two series of experiments (pH 6 and pH 4.5) can be seen in Figure 4. The results of graphical analysis of plots in Figure 4 suggest the existence of two types of binding sites. The steep part of the graph is due to the specific binding sites with high affinity from Eu3+, while the “gentle” part describes the nonspecific binding. Scatchard plots for the experiments at both pH values (pH 6 and 4.5) indicate the same two-stage character of binding.

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DISCUSSION Eu binding by BSA molecules was investigated using the fluorescence quenching technique, when the intrinsic fluorescence of protein is measured at different concentrations of quencher. The second approach deals with the luminescence of Eu3+ as an alternative indicator of interaction processes. The third approach allowed the concentration of free and bound ions to be obtained from the ratio of peak intensities in the Eu3+ spectrum and the analysis of experimental data to be performed using the standard procedure based on the Scatchard plot. The n and Keq values obtained using all three of these approaches are provided in Table 1. The number of binding sites (n = 1 at both pH values) and equilibrium constants (log Keq ∼ 3) determined from the quenching of BSA intrinsic fluorescence using the modified Stern−Volmer equation (eq 2) are lower than the corresponding values obtained from Eu3+ luminescence measurements. Moreover, n and Keq determined with FQT are independent of the pH of the sample, while the I594/I616 curves (Figure 3B) and the Scatchard plots (Figure 4) indicate the decrease of both n and Keq at pH 4.5. The following parameters for lanthanide binding by albumin can be found in the literature. In the article of Reuben,38 where the number of binding sites for Gd3+ and equilibrium constants for Gd3+−human serum albumin (HSA) complexes were determined from the water proton relaxation rates, the values 3+

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ligand binding in derivation of the modified Stern−Volmer equation led to “infinite cooperativity” and should be considered unreliable. Here, we performed the numerical modeling of the binding process and fluorescence quenching curves based on the model of independent sites. If the model of complex formation is known, the distribution of protein−metal complexes on ligand concentration can be obtained, and then, using a set of fluorescence quantum yields, the fluorescence quenching curves can be calculated and their slopes on a double logarithmic scale can be determined. If the binding sites can be regarded as independent and are characterized by equal affinities for ligands, the complexation process can be expressed with the following equations:

F=

i=0

(4)

where [S], [Q], and [SQ] are the concentrations of free binding sites, ligand, and occupied sites, respectively. Next, to obtain the concentration of protein complex with k ligands and assuming that each protein obtains n binding sites, one should calculate all possible combinations of ligand placement taking into account the fact of independence of the binding process for each site [PQ k] =

⎛n ⎞ k n−k ⎜ ⎟p (1 − p) [P]T ⎝k ⎠

F [P] = F0 [P]T

(5)

Keq[Q] [SQ] = n[P]T 1 + Keq[Q]

(8)

As it is stated in a number of works, the relevancy of static quenching of protein fluorescence on ligand binding is doubtful. For instance, it can be seen in Figure 5 which presents complex distribution on [Q], that at ligand concentration [Q] ∼ 10−4 M free protein molecules are almost absent. Thus, according to eq 8, a dramatic decrease of fluorescence intensity should be observed in the experiment. At the same time, fluorescence decrease upon addition of such ligand concentration is less than 40% for the majority of FQT experiments and less than 10% for our experiment (see Figure 2A). Moreover, the larger the equilibrium constant for the first binding site (e.g., the typical log Keq values can exceed 6 for specific binding40,41), the larger the decrease that should be observed. Figure 6 demonstrates a fluorescence quenching curve on a double logarithmic scale calculated on the basis of distributions that are shown in Figure 5 and eq 8 under the assumption of static fluorescence quenching (i.e., the quantum yields for PQ1−PQ5 complexes are equal to zero).

where [P]T is the total concentration of protein in the system and p is the probability of binding site occupation given by p≡

(7)

where αi is the fluorescence quantum yield of the complex with i ligands and n is the number of all binding sites. In this expression, no assumptions about the fluorescence quenching mechanism or the complexation model are made. The use of independent sites grouping into molecules given by eqs 5 and 6 allows one to take into account the hierarchy of sites by the selection of specific sets of quantum yields in eq 7, e.g., no quenching for high-affinity sites and strong quenching for nonspecific sites of the binding cooperativity. We performed the modeling of the fluorescence quenching curves for the cases of static and dynamic quenching. Static Quenching. Using the complex concentrations [PQi] that can be obtained from eqs 4−6, one can calculate fluorescence intensity at certain ligand concentration [Q] if the set of αi is known. The double logarithmic Stern−Volmer equation (eq 2) is derived under the assumption of complete fluorescence quenching for a molecule complexed with at least one ligand, i.e., under the assumption of static quenching. Hence, fluorescence intensity is assumed to be proportional to the concentration of free protein in the system:

S + Q ⇌ SQ [SQ] = Keq[S][Q]

∑ αi[PQ i]

(6)

The modeling results for n = 5, log Keq = 4 are presented in Figure 5.

Figure 5. Distribution of protein−metal complexes calculated using the model of independent sites for [P]T = 10−5 M, n = 5, log Keq = 4.

Here, for simplicity, we consider the system characterized by the single type of binding sites. The behavior of the modeled system remains the same if the number of binding site types is changed. Using the calculated distribution of protein−metal complexes on the metal’s concentrations, the numerical modeling of the fluorescence quenching curve can be performed. The general expression for protein intrinsic fluorescence intensity F dependence on [Q] is given by

Figure 6. Double logarithmic Stern−Volmer plot calculated under the assumption of static quenching of fluorescence for n = 5, log Keq = 4. 6630

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fluorescent binding sites. The steep part of this graph is also never observed in experiments. This can be explained by residual fluorescence (unreliability of the static quenching model). Finally, the use of a fluorescence probe (in our case, Eu3+) lacks the limitations typical for the fluorescence quenching technique and is capable of providing a reliable description of ligand binding.

Linear approximation of the gentle part of the fluorescence quenching curve ([Q] ≲ 10−4 M) gives n values close to unity, despite the fact that the modeling was performed for n = 5. The steep part of the presented curve (case of high quencher concentrations, [Q] ≳ 10−4 M) gives n that is equal to the number of nonfluorescent complexes, that was put in the model (in our case, n = 5). Dynamic Quenching. To calculate the fluorescence quenching curve in the case of dynamic quenching of fluorescence, a set of fluorescence quantum yields αi (see eq 7) must be introduced. Assuming the fluorescence quantum yield of free protein molecule to be unity, we supposed that occupation of each site leads to the equal subsequent 5% decrease of αi. We note that any other reasonable set of αi will lead to the same results; the only condition is the monotonous decrease of fluorescence quantum yield with increase of the number of bound ligands i. The fluorescence quenching curve modeled for the complex distribution shown in Figure 5 and this αi set is presented in Figure 7.

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CONCLUSION We made use of fluorescence spectroscopy to investigate the interaction between bovine serum albumin and Eu3+ ions in aqueous solution at two pH values which are below (pH 4.5) and above (pH 6.0) the pKa for this protein. Fluorescence is known to be widely used to study ligand binding, though its limitations and pitfalls were presented in the literature. The system studied in this work included blood plasma transport protein BSA and heavy metal ion Eu3+ which is a classical luminescent probe. The literature data strongly suggested that in this case several binding sites with different affinities were expected for BSA; moreover, Eu3+ binding should be significantly weaker at pH 4.5 compared to pH 6.0 if electrostatic interaction is essential for complex formation. We performed the standard procedures applied for the assessment of ligand binding parameters which included the measurement of intrinsic protein fluorescence quenching on ligand addition to solution, processing of the quenching data using the Stern− Volmer and double logarithmic Stern−Volmer equations, and the measurement of ligand florescence upon binding. The latter allowed the number of free and bound ligands to be determined and data to be represented in the form of a Scatchard plot. It was shown that, while Eu3+ luminescence data provided an adequate number of binding sites at pH 6 (n1 = 2, n2 = 4) and indicated a reasonable decrease of binding affinity at pH 4.5 (n1 = 1, n2 = 2), the double logarithmic Stern−Volmer equation gave n = 1 for both pH values. To further investigate the observed discordance, we performed numerical modeling of quenching curves for the cases of static and dynamic quenching. Using the model of similar independent binding sites, the distributions of protein−ligand complexes at different ligand concentrations were calculated, and then, the fluorescence quenching curves were plotted using certain sets of fluorescence quantum yields. The results of the modeling provided the slope of fluorescence quenching curves on a double logarithmic scale that was close to unity and did not depend on the initial number of binding sites used for numerical modeling. We also provided an analytical description of this fact. It can be concluded that one should use intrinsic fluorescence quenching of proteins to assess ligand binding parameters with extreme caution, and the use of alternative indicators (e.g., ligand’s fluorescence) is strongly recommended to avoid artifacts.

Figure 7. Double logarithmic Stern−Volmer plot calculated under the assumption of dynamic quenching of fluorescence for n = 5, log Keq = 4.

Here, the linear approximation of the “steep” part of the presented curve ([Q] ≲ 10−4 M) gives n = 1, despite the fact that a value of binding sites of n = 5 was used. Linear approximation of the “gentle” part of the curve ([Q] > 10−4 M) gives n that is equal to 0, as at these concentrations of ligands all protein binding sites are occupied and the increase of ligand concentration in the solution would not influence fluorescence intensity. We note that the described trends hold true for any arbitrary value of n. The plots calculated in the case of static and dynamic quenching for n = 1, 3, 5, and 7, their approximation, and further analytical description are presented in Appendix B in the Supporting Information (Figures SI1 and SI2). As a result, in both cases of static and dynamic quenching, the approximation of the initial part (low concentrations of quencher) of fluorescence quenching curves on a double logarithmic scale gives n = 1. In the case of dynamic quenching, the saturation of the double logarithmic Stern−Volmer plot can be observed (Figure 7), while we did not find such dependencies in the literature. The possible reason could be that the increase of ligand concentration leads to the protein conformational changes and the appearance of new binding sites, which leads to the further fluorescence intensity decrease.44 In the case of high quencher concentrations for the static quenching, the linear approximation of the modeled “log−log” Stern−Volmer plot provides the number of non-

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ASSOCIATED CONTENT

* Supporting Information S

The deconvolution procedure for the determination of free and bound europium(III) concentrations is described in Appendix A. Analytical description of the fluorescence quenching curves for the cases of static and dynamic quenching as well as the modeling results are presented in Appendix B. This material is available free of charge via the Internet at http://pubs.acs.org. 6631

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(18) Lu, X.-L.; Fan, J.-J.; Liu, Y.; A.-X, H. Characterization of the Interaction Between Cationic Erbium (III) - Porphyrin Complex with Bovine Serum Albumin. J. Mol. Struct. 2009, 934, 1−8. (19) Krause-Heuer, A. M.; Price, W. S.; Aldrich-Wright, J. R. Spectroscopic Investigations on the Interactions of Potent Platinum(II) Anticancer Agents with Bovine Serum Albumin. J. Chem. Biol. 2012, 5, 105−113. (20) Crouse, H. F.; Potoma, J.; Nejrabi, F.; Snyder, D. L.; Chohan, B. S.; Basu, S. Quenching of Tryptophan Fluorescence in Various Proteins by a Series of Small Nickel Complexes. Dalton Trans 2012, 41, 2720−2731. (21) Shen, L.; Yang, Z.; Tang, R. Synthesis, Luminescence Properties of Eu(III) and Tb(III) Complexeswith a Novel Aromatic Carboxylic Acid and Their Interactions with Bovine Serum Albumin. Spectrochim. Acta, Part A 2012, 98, 170−177. (22) Yang, Z.; Tang, R.; Zhang, Z. Synthesis and Luminescence Properties of Tb(III) Complex with a Novel Pyrazolone Ligand and Its Interactions with Bovine Serum Albumin. J. Mol. Struct. 2012, 1030, 19−25. (23) Lissi, E.; Abuin, E. On the Evaluation of the Number of Binding Sites in Protein from Steady State Fluorescence Measurements. J. Fluoresc. 2011, 21, 1831−1833. (24) Tang, L.; Jia, W. A Comparison Study on the Binding of Hesperetin and Luteolin to Bovine Serum Albumin by Spectroscopy. Spectrochim. Acta, Part A 2013, 103, 114−119. (25) Fasman, G. D. Practical Handbook of Biochemistry and Molecular Biology; CRC Press: Boca Raton, USA, 1989. (26) Pfefferle, J.-M.; Bunzli, J.-C. G. Interaction Between the Buffer Tris and Eu(III) Ion: Luminescence and Potentiometric Investigation. Helv. Chim. Acta 1989, 72, 1487−1494. (27) Hanlon, D. P.; Watt, D. S.; Westhead, E. W. The Interaction of Divalent Metal Ions with Tris Buffer in Dilute Solution. Anal. Biochem. 1966, 16, 225−233. (28) Sokolowska, M.; Pawlas, K.; Bal, W. Effect of Common Buffers and Heterocyclic Ligands on the Binding of Cu(II) at the Multimetal Binding Site in Human Serum Albumin. Bioinorg. Chem. Appl. 2010, 2010, 725153 1−7. (29) Fischer, B. E.; Haring, U. K.; Tribolet, R.; Sigel, H. Metal Ion/ Buffer Interactions. Stability of Binary and Ternary Complexes Containing 2-amino-2(hydroxymethyl)-1,3-propanediol (tris) and Adenosine 5′-triphosphate (atp). Eur. J. Biochem. 1979, 94, 523−530. (30) Bohme, U.; Scheler, U. Effective Charge of Bovine Serum Albumin Determined by Electrophoresis NMR. Chem. Phys. Lett. 2007, 435, 342−345. (31) Kragh-Hansen, U. Molecular Aspects of Ligand Binding to Serum Albumin. Pharmacol. Rev. 1981, 33, 17−53. (32) Turro, C.; Fu, P. K.-L.; Bradley, P. Lanthanide Ions as Luminescent Probes of Proteins and Nucleic Acids. Met. Ions Biol. Syst. 2003, 40, 323−353. (33) Werts, M.; Jukes, R.; Verhoeven, J. The Emission Spectrum and Radiative Lifetime of Eu3+ in Luminescent Lanthanide Complexes. Phys. Chem. Chem. Phys. 2002, 4, 1542−1548. (34) Richardson, F. S. Terbium(III) and Europium(III) Ions As Luminescent Probes and Stains for Biomolecular Systems. Chem. Rev. 1982, 82, 541−552. (35) Görller-Walrand, C.; Binnemans, K. Spectral Intensities of f-f Transitions. Handb. Phys. Chem. Rare Earths 1998, 25, 101−264. (36) Scatchard, G. The Attractions of Proteins for Small Molecules and Ions. Ann. N.Y. Acad. Sci. 1949, 61, 660−672. (37) Saboury, A. Application of a New Method for Data Analysis of Isothermal Titration Calorimetry in the Interaction Between Human Serum Albumin and Ni2+. J. Chem. Thermodyn. 2003, 35, 1975−1981. (38) Reuben, J. Gadolinium (III) As a Paramagnetic Probe for Proton Relaxation Studies of Biological macromolecules.Binding to Bovine Serum Albumin. Biochemistry 1971, 10, 2834−2838. (39) Schomacker, K.; Mocker, D.; Munze, R.; Beyer, G.-J. Stabilities of Lanthanide-Protein Complexes. Int. J. Radiat. Appl. Instrum., Part A 1988, 39, 261−264.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The reported study was supported by Russian Scientific Foundation (grant 14-15-00602) and Russian Foundation for Basic research (projects 14-02-31814 and 14-03-31723).

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REFERENCES

(1) Kodama, K. Studies on the Interaction Between Heavy Metal Salt and Protein. J. Biochem. 1923, 2, 505−524. (2) Cohn, E.; Surgenor, D.; Schmid, K.; Batchelor, W.; Isliker, H.; Alameri, E. The Interaction of Plasma Proteins with Heavy Metals and with Alkaline Earths, with Specific Anions and Specific Steroids, with Specific Polysaccharides and with the Formed Elements of the Blood. Discuss. Faraday Soc. 1953, 13, 176−189. (3) Pyle, A.; Barton, J. K. Probing Nucleic Acids with Transition Metal Complex. Prog. Inorg. Chem. 1990, 38, 413−476. (4) Shiman, R.; Draper, D. Stabilization of RNA Tertiary Structure by Monovalent Cations. J. Mol. Biol. 2000, 302, 79−91. (5) Peng, M.; Shi, S.; Zhang, Y. Influence of Cd2+, Hg2+ and Pb2+ on (+)-catechin Binding to Bovine Serum Albumin Studied by Fluorescence Spectroscopic Methods. Spectrochim. Acta, Part A 2012, 85, 190−197. (6) Liu, Y.; Zhang, L.; Liu, R.; Zhang, P. Spectroscopic Identification of Interactions of Pb2+ with Bovine Serum Albumin. J. Fluoresc. 2012, 22, 239−245. (7) Bush, A. Metals and Neuroscience. Curr. Opin. Chem. Biol. 2000, 4, 184−191. (8) Petrova, G.; Petrusevich, Y.; Evseevicheva, A. Molecular Clusters in Water Protein Solutions in the Presence of Heavy Metal Ions. Gen. Physiol. Biophys. 1998, 17, 97−104. (9) Uversky, V.; Li, J.; Fink, A. Metal-triggered Structural Transformations, Aggregation, and Fibrillation of Human α-synuclein. A Possible Molecular Link Between Parkinson’s Disease and Heavy Metal Exposure. J. Biol. Chem. 2001, 276, 44284−44296. (10) Kazakov, V. P.; Ostakhov, S. S.; Farrakhova, G. G. Photoinduced Electron Transfer from Higher Excited Singlet States of Tryprophan: 1. Effects of the Excitation Wavelength, pH, and Temperature on Quenching of Tryptophan Fluorescence by Europium(III) Ions. High Energy Chem. 2008, 42, 283−286. (11) Wu, L.-Z.; Ma, B.-L.; Zou, D.-W.; Tie, Z.-X.; Wang, J.; Wang, W. Influence of Metal Ions on Folding Pathway and Conformational Stability of Bovine Serum Albumin. J. Mol. Struct. 2008, 877, 44−49. (12) Jackson, G.; Murray, I.; Hosszu, L.; Gibbs, N.; Waltho, J.; Clarke, A.; Collinge, J. Location and Properties of Metal-binding Sites on the Human Prion Protein. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8531−8535. (13) Belatik, A.; Hotchandani, S.; Carpentier, R.; Tajmir-Riahi, H.-A. Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins. PLoS One 2012, 7, 1−9. (14) Masuoka, J.; Hegenauer, J.; Van Dyke, B.; Saltman, P. Intrinsic Stoichiometric Equilibrium Constants for the Binding of Zinc(II) and Copper(II) to the High Affinity Site of Serum Albumin. J. Biol. Chem. 1993, 268, 21533−21537. (15) Ohyoshi, E.; Hamada, Y.; Nakata, K.; Kohata, S. The Interaction Between Human and Bovine Serum Albumin and Zinc Studied by a Competitive Spectrophotometry. J. Inorg. Biochem. 1999, 75, 213−218. (16) Sadler, P. J.; Viles, J. H. 1H and 113Cd NMR Investigations of Cd2+ and Zn2+ Binding Sites on Serum Albumin: Competition with Ca2+, Ni2+, Cu2+, and Zn2+. Inorg. Chem. 1996, 35, 4490−4496. (17) Lakowicz, J. Principles of Fluorescence Spectroscopy; Springer: New York, 2006. 6632

dx.doi.org/10.1021/jp501277z | J. Phys. Chem. B 2014, 118, 6626−6633

The Journal of Physical Chemistry B

Article

(40) Ganjali, M.; Faridbod, F.; Saboury, A.; Davarkhah, N.; Divsalar, A.; Rezaei Behbehani, G.; Norouzi, P. A New Approach for Protein Binding Isotherm Analysis by Means of an Electrochemical Microsensor. Int. J. Electrochem. Sci. 2010, 5, 630−638. (41) Rezaei Behbehani, G.; Divsalar, A.; Saboury, A.; Faridbod, F.; Ganjali, M. A High Performance Method for Thermodynamic Study on the Binding of Human Serum Albumin with Erbium Chloride. J. Therm. Anal. Calorim. 2009, 96, 663−668. (42) Li, X.; Wang, C.; Li, J.; Wang, Z. Microcalorimetric Studies on the Interactions of Lanthanide Ions with Bovine Serum Albumin. J. Therm. Anal. Calorim. 2007, 89, 899−905. (43) Van de Weert, M.; Stella, L. Fluorescence Quenching and Ligand Binding: A Critical Discussion of a Popular Methodology. J. Mol. Struct. 2011, 998, 144−150. (44) Anand, U.; Jash, C.; Mukherjee, S. Spectroscopic Probing of the Microenvironment in a Protein- Surfactant Assembly. J. Phys. Chem. B 2010, 114, 15839−15845.

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