Interaction of procyanidin B3 with bovine serum albumin

Abstract

Proanthocyanidins are a mixture of monomers, oligomers, and polymers of flavan-3-ols that are widely distributed in the plant kingdom. One of the most widely studied proanthocyanidins is procyanidin B3. In this study, the interaction between procyanidin B3 and bovine serum albumin (BSA) was investigated using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy, UV-vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular docking. Thermodynamic investigations reveal that the electrostatic interaction and hydrophobic interaction are the major binding forces in the binding of procyanidin B3 to BSA. The binding of procyanidin B3 to BSA is synergistically driven by enthalpy and entropy. Fluorescence experiments suggest that procyanidin B3 can quench the fluorescence of BSA through a static quenching mechanism. The obtained binding constants and the equilibrium fraction of unbound procyanidin B3 show that procyanidin B3 can be stored and transported from the circulatory system to reach its target organ. Binding site I is found to be the primary binding site for procyanidin B3, which is consistent with the result of molecular docking studies. Additionally, as shown by the UV-vis absorption, synchronous fluorescence spectroscopy, FT-IR and CD, procyanidin B3 may induce conformational and microenvironmental changes of BSA.

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1. Introduction

Proanthocyanidins are ubiquitous and present as the second most abundant group of natural phenolics after lignin.¹ They are polymers of flavan-3-ols which present a wide variety of chemical structures with different features: (1) among their structural units there may be (epi)catechin, (epi)gallocatechin or (epi)afzelechin moieties, in which case they are called procyanidins, prodelphinidins and propelargonidins, respectively; (2) the linkage between the different units of the chain may be a B-type linkage (C–C bond between monomers) or an A-type linkage (containing an ether bond in addition to the C–C bond); (3) there may be galloyl groups associated with some of the moieties in the chain.² The widespread presence of proanthocyanidins in plants makes them an important part of the human diet, as they are found in fruits, berries, beans, nuts, cocoa, and wine.³ Besides their participation in food quality attributes such as astringency, bitterness, aroma and color formation,⁴ proanthocyanidin consumption has been associated with numerous health benefits due their antioxidant, vasodilatory, anticarcinogenic, antiallergic, anti-inflammatory, antibacterial, cardioprotective, immune-stimulating, anti-viral and estrogenic activities. Furthermore, proanthocyanidins have been reported to inhibit lipid peroxidation, platelet aggregation, capillary permeability and fragility. They also modulate the activity of enzyme systems including phospholipase A₂, cyclooxygenase and lipoxygenase.⁵ So they are considered as functional ingredients in botanical and nutritional supplements.

Proanthocyanidins have the ability to interact with proteins that make them worthy of attention by diverse areas such as medicine, toxicology, chemistry, food science, and agriculture. The binding study between proanthocyanidins and protein had been done previously.^6–9 However, to our knowledge, an accurate and full basic data for clarifying the binding mechanisms of proanthocyanidins to plasma proteins remain unclear. Serum albumin is the most abundant protein in blood plasma (∼60%) and serves as a depot and transport protein for numerous endogenous and exogenous compounds.¹⁰ Knowledge of interaction mechanisms between proanthocyanidins and serum albumin are very important for us to understand the pharmacokinetics and pharmacodynamics of proanthocyanidins. First, the proanthocyanidins–HSA interaction plays a dominant role in the bioavailability of proanthocyanidins because the bound fraction of proanthocyanidins is a depot, whereas the free fraction of proanthocyanidins shows pharmacological effects.¹¹ In addition, if proanthocyanidins are metabolized and excreted from the body too fast because of low protein binding, proanthocyanidins won't be able to provide their therapeutic effects. Alternatively, if proanthocyanidins have high protein binding and are metabolized and excreted too slowly, it may increase the half-life of proanthocyanidins in vivo and lead to undesired side effects.¹² Furthermore, very high affinity binding of proanthocyanidins to serum albumin may prevent proanthocyanidins from reaching the target at all, resulting in insufficient tissue distribution and efficacy. In a word, the absorption, distribution, metabolism, and excretion properties of proanthocyanidins can be significantly affected as a result of their binding to serum albumin. Besides, there is evidence of conformational changes of serum albumin induced by its interaction with drugs, which may affect serum albumin's biological function as the carrier protein.¹³ Consequently, investigation of the binding of proanthocyanidins to serum albumin is of great importance. As the sequences of human serum albumin (HSA) and BSA are 76% conserved, BSA is commonly substituted for HSA in experiments due to its availability and lower cost.¹⁴

One of the most widely studied proanthocyanidins is procyanidin B3 (catechin-(4β → 8)-catechin; molecular structure: inset of Fig. 1) due to its high abundance in the human diet and relevant antioxidant activity.⁹ In the present work, a comprehensive investigation was performed for the binding properties of procyanidin B3 to BSA under the physiological conditions. Using isothermal titration calorimetry (ITC), in combination with different spectroscopic methods and molecular docking, the binding information, including thermodynamic parameters, quenching mechanism, binding parameters, the equilibrium fraction of unbound procyanidin B3, high-affinity binding site, and conformation changes of BSA was investigated. The study provides an accurate and full basic data for clarifying the binding mechanism of procyanidin B3 with BSA and is helpful for understanding its effect on protein function during the blood transportation process and its biological activity in vivo.

Fig. 1 (A) Raw data for the titration of 7 × 10⁻³ mol L⁻¹ procyanidin B3 with 6 × 10⁻⁴ mol L⁻¹ BSA at pH 7.40 and 298 K, showing the calorimetric response as successive injections of procyanidin B3 are added to the sample cell. (B) Integrated heat profile of the calorimetric titration shown in panel A. The solid line represents the best nonlinear least-squares fit to the independent binding sites model.

2. Experimental

2.1. Materials

BSA, procyanidin B3, warfarin, and ibuprofen were purchased from Sigma-Aldrich Chemicals Company (USA). Procyanidin B3 was directly dissolved in phosphate buffer solution of pH 7.40 (0.01 mol L⁻¹ PBS). The stock solution of procyanidin B3 was prepared before use and did not keep this solution for too long after prepared because its strong reduction property. The water used to prepare the solution was double distilled water. The BSA was dissolved in a phosphate buffer solution of pH 7.40 (0.01 mol L⁻¹ PBS). The concentration of the BSA was determined on a TU-1810 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China) using the extinction coefficient ε₂₈₀ = 44 720 mol⁻¹ L cm⁻¹.¹⁵ The reported pH determined on a PHS-2C pH-meter (Shanghai DaPu Instruments Co., Ltd, Shanghai, China) at ambient temperature. Sample masses were accurately weighed on a microbalance (Sartorius, BP211D) with a resolution of 0.01 mg. All other reagents were all of analytical reagent grade and were used as purchased without further purification.

2.2. Isothermal titration calorimetry (ITC)

Titration of BSA with procyanidin B3 was performed using a Model Nano-ITC 2G biocalorimetry instrument (TA, USA) at 298 K. All these solutions were thoroughly degassed prior to the titrations to avoid the formation of bubbles in the calorimeter cell. The sample cell was loaded with the phosphate buffer (PBS, 0.01 mol L⁻¹) or protein solution and the reference cell contained double distilled water. In a typical experiment, buffered BSA solution was placed in the 950 μL sample cell of the calorimeter and procyanidin B3 solution was loaded into the injection syringe. Injections were started after baseline stability had been achieved. Procyanidin B3 was titrated into the sample cell by means of syringes via 25 individual injections, the amount of each injection was 10 μL. The first injection of 10 μL was ignored in the final data analysis. The contents of the sample cell were stirred throughout the experiment at 200 rpm to ensure thorough mixing. Raw data were obtained as a plot of heat (μJ) against injection number and featured a series of peaks for each injection. These raw data peaks were transformed using the instrument's software to obtain a plot of enthalpy change per mole of injectant (ΔH⁰, kJ mol⁻¹) against molar ratio. Control experiments included the titration of procyanidin B3 solution into buffer, buffer into BSA, and buffer into buffer, controls were repeated for the same BSA concentration used. The last two controls resulted in small and equal enthalpy changes for each successive injection of buffer and, therefore, were not further considered in the data analysis.¹⁶ Corrected data refer to experimental data after subtraction of the procyanidin B3 into buffer control data. Estimated binding parameters were obtained from ITC data using NanoAnalyze software provided by the manufacturer. Fitting the data according to the independent binding model resulted in the stoichiometry of binding (n), the equilibrium binding constant (K), and enthalpy of complex formation (ΔH⁰). The standard changes in free energy (ΔG⁰) and entropy (ΔS⁰) are calculated using the following equations:¹⁷

ΔG⁰ = −RT ln K (1)

ΔG⁰ = ΔH⁰ − TΔS⁰ (2)

2.3. Fluorescence measurements

The fluorescence measurements were performed on Cary Eclipse fluorescence spectrophotometer (Varian, USA) equipped with a 1.0 cm quartz cell and a thermostat bath. The BSA concentration was kept at 2 × 10⁻⁶ mol L⁻¹. The excitation and emission slit widths were fixed at 5 nm. The excitation wavelength was set at 280 nm (excitation of the Trp and Tyr), and the emission spectra were read at 300–450 nm at a scan rate of 100 nm min⁻¹. The synchronous fluorescence spectra were scanned from 280 to 330 nm (Δλ = 15 nm) and from 310 to 380 nm (Δλ = 60 nm), respectively. In the site marker competitive experiment, the procyanidin B3 was gradually added to the solution of BSA and site markers held in equimolar concentrations (2 × 10⁻⁶ mol L⁻¹). The site markers used were warfarin for site I and ibuprofen for site II.

The fluorescence measurements are hindered by the inner-filter effect, which is that small ligands absorb the light at the excitation and emission wavelengths of proteins and leads to unreliable results.¹⁸ Thus it is very important to subtract such an effect from the raw quenching data. The extent of this effect can be roughly estimated with the following equation:¹⁹

F_cor = F_obsd10^{(A_ex+A_em)/2} (3)

where F_cor and F_obsd are the corrected and observed fluorescence intensities, respectively, whereas A_ex and A_em are the sum of the absorbance of protein and ligand at the excitation and emission wavelengths, respectively. The fluorescence intensity utilized in this study is the corrected intensity.

2.4. Absorbance measurements

UV-vis absorption spectra were recorded with a TU-1810 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China) equipped with 1.0 cm quartz cells at 298 K. The solutions of the blank buffer and sample were placed in the reference and sample cuvettes, respectively.

2.5. Fourier transform infrared (FT-IR) measurements

FT-IR measurements were performed on an Avatar 360 E. S. P. FT-IR spectrometer (PerkinElmer). All spectra were taken via the ATR method with a resolution of 4 cm⁻¹. The FT-IR spectra of BSA (1.0 × 10⁻⁴ mol L⁻¹) in the absence and presence of procyanidin B3 were recorded in the range of 4000–700 cm⁻¹ at pH 7.40 phosphate buffer and room temperature. The molar ratio of procyanidin B3 to BSA was maintained at 1 : 10. The corresponding absorbance contribution of buffer and free procyanidin B3 solution was recorded and digitally subtracted at the same condition. The secondary structure compositions of free BSA and its procyanidin B3 complex were estimated by the FT-IR spectra and the curve-fitted results of the amide I band.

2.6. Circular dichroism (CD) measurements

The CD measurements were carried out on a Jasco J-715 spectropolarimeter under constant nitrogen flush. For measurements in the far-UV region (190–260 nm), a quartz cell with a path length of 0.2 cm was used. Three scans were accumulated with continuous scan mode and a scan speed of 200 nm min⁻¹ with data being collected at 0.2 nm and response time of 2 s. The sample temperature was maintained at 298 K. The protein concentration was fixed to 2.0 × 10⁻⁶ mol L⁻¹ and the procyanidin B3 concentrations used were 0, 2.0, 4.0, and 8.0 × 10⁻⁵ mol L⁻¹ in phosphate buffer (PBS, 0.01 mol L⁻¹), pH 7.40. All observed CD spectra were baseline subtracted for phosphate buffer (pH 7.40), and results were taken as CD ellipticity in mdeg.

2.7. Molecular docking study

As the crystal structure of BSA is unavailable in Protein Data Bank,^20,21 so a homology model was used for the docking with procyanidin B3. The crystal structure of BSA was modeled with Modeller_9v7. The sequence of the protein was obtained from NCBI database (http://www.ncbi.nlm.nih.gov/Database/; ID: CAA76847). The Basic Local Alignment Search Tool (BLAST) of the sequence of BSA and HSA (PDB database) had given the highest homogeneous identity crystal structure of HSA (PDB entry: 1AO6). The model structure of BSA used here was according to this crystal structure. The potential of the 3D structure of BSA was assigned according the Amber 4.0 force field with Kollman-all-atom charges. The initial structure of all the molecules was generated by molecular modeling software Sybyl 6.9.1. The geometries of procyanidin B3 was subsequently optimized using the Tripos force field with Gasteiger–Hückel charges with a gradient of 0.005 kcal mol⁻¹. FlexX program was applied to calculate the possible conformation of the ligand that binds to the protein. During docking process, a maximum of 10 conformers was considered for procyanidin B3. The conformer with the lowest binding free energy was used for further analysis. As is addressed above, a computational model of the target receptor was built. The output from Sybyl was rendered with PyMol.

3. Results and discussion

3.1. Isothermal titration calorimetry (ITC) studies

Isothermal titration calorimetry (ITC), which measures directly the heat evolved during a reaction, is the method of choice for obtaining thermodynamic information. This is because only ITC allows researchers to obtain directly the variations of enthalpy ΔH⁰ and of entropy ΔS⁰, as well as the association constant K and the stoichiometry of binding n, for an association process.²²

A representative calorimetric titration profile of 7 × 10⁻³ mol L⁻¹ procyanidin B3 with 6 × 10⁻⁴ mol L⁻¹ BSA at pH 7.40 and 298 K is shown in Fig. 1A. The thermodynamic parameters for the interaction of procyanidin B3 with BSA obtained from ITC are listed in Table 1. Each peak in the binding isotherm represents a single injection of the procyanidin B3 into the BSA solution. The exothermicity of the calorimetry peaks in Fig. 1A is believed to be due to the strong interaction between procyanidin B3 and BSA. As the sites available on BSA become progressively occupied during titration, the exothermicity of the peaks decreases and eventually saturates. The weak endothermic response that is more clearly observed after saturation of BSA with procyanidin B3 is believed to arise due to breaking of intermolecular bonds in the procyanidin B3 as a consequence of dilution during titration.^23–26 Fig. 1B shows the plot of enthalpy change (ΔH⁰) against [procyanidin B3]/[BSA] molar ratio. The solid smooth line in Fig. 1B represents the best fit of the experimental data using the independent binding sites model with the stoichiometric binding number (n), association constant (K), and enthalpy change (ΔH⁰) of 1.008, 2.183 × 10⁴ mol⁻¹ L and −15.696 kJ mol⁻¹, respectively. The free energy change (ΔG⁰) and the entropy change (ΔS⁰) evaluated from eqn (1) and (2) is −24.754 kJ mol⁻¹ and 30.396 J mol⁻¹ K⁻¹, respectively.

Table 1 Thermodynamic parameters for the interaction of procyanidin B3 with BSA obtained from ITC at 298 K and pH 7.40

T (K)	K (L mol^−1)	n	ΔH^0 (kJ mol^−1)	ΔG^0 (kJ mol^−1)	ΔS^0 (J mol^−1 K^−1)
298	2.183 × 10^4	1.008	−15.696	−24.754	30.396

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The association constant (K) between procyanidin B3 and BSA is moderate compared to other strong protein–ligand complexes with binding constants ranging from 10⁷ to 10⁸ L mol⁻¹.²⁷ From the point of pharmacokinetics, the moderate affinity of procyanidin B3 for BSA leads to a faster diffusion rate in the circulatory system to reach target site.²⁸ The value of the stoichiometric binding number n approximately equals to 1, suggesting that one molecule of procyanidin B3 combines with one molecule of BSA and no more procyanidin B3 binding to BSA occurs at concentration ranges used in this study. The negative values of free energy (ΔG⁰) and enthalpy (ΔH⁰) support that the binding of procyanidin B3 to BSA is spontaneous and exothermic. Ross and Subramanian²⁹ have characterized the sign and magnitude of the thermodynamic parameter associated with various individual kinds of interaction which may take place in protein association process. From the standpoint of water structure, the positive entropy (ΔS⁰) is often regarded as a typical signature of hydrophobic interaction, since the water molecules which are arranged in an orderly fashion around the ligand and the protein molecules acquire a more random configuration as a result of hydrophobic interaction.³⁰ There are several types of weak interactions simultaneously contribute to the negative enthalpy (ΔH⁰) values for the procyanidin B3–BSA system. (i) When a procyanidin B3 molecule partially inserts itself into a hydrophobic cavity of BSA molecule formed by folding and twisting of peptide chain, the hydrophobic interaction between the procyanidin B3 molecule and the cavity would cause an exothermic process.³¹ (ii) The expulsion of water molecules from the hydrophobic cavity to bulk solution is also exothermic,³² which leads to the negative heat effect more evident, because energy of water molecules in hydrophobic microenvironments is more than that in the bulk aqueous solution. (iii) Directly electrostatic interaction of dipole groups of the procyanidin B3 molecules with peptide sections of BSA molecules also causes exothermic effect.³³ Therefore, the electrostatic interaction and hydrophobic interaction are the major binding forces in the binding of procyanidin B3 to BSA. By the eqn (2), the change of Gibbs free energy (ΔG⁰) is the comprehensive embodiment of the changes of enthalpy (ΔH⁰) and entropy (ΔS⁰). The binding of procyanidin B3 to BSA is synergistically driven by enthalpy and entropy.

3.2. Fluorescence spectroscopic studies

3.2.1. Effect of procyanidin B3 on BSA fluorescence. Fig. 2 shows the fluorescence emission spectra obtained for BSA at pH 7.40 with the addition of procyanidin B3. It can be seen that the fluorescence intensity of BSA decreased in the presence of procyanidin B3, indicating that the binding of procyanidin B3 to BSA quenched the intrinsic fluorescence of BSA.³⁴ Furthermore, a small blue shift (from 346 to 344 nm) is observed with increasing procyanidin B3 concentration, which suggests that the fluorophores of BSA is placed in a more hydrophobic environment after the addition of procyanidin B3.³⁵

Fig. 2 Emission spectra of BSA in the presence of different concentrations of procyanidin B3 at 298 K and pH 7.40. c(BSA) = 2 × 10⁻⁶ mol L⁻¹; c(procyanidin B3)/10⁻⁵ mol L⁻¹, (a–j): 0; 0.2; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0. The inset corresponds to the molecular structure of procyanidin B3.

3.2.2. Fluorescence quenching mechanisms. The different mechanisms of quenching are usually classified as either dynamic quenching or static quenching. For fluorescence quenching, the decrease in intensity is usually described by the Stern–Volmer equation:³⁶

F₀/F = 1 + k_qτ₀[Q] = 1 + K_SV[Q] (4)

where F₀ and F represent the steady-state fluorescence intensities in the absence and presence of quencher, respectively. k_q is the bimolecular quenching constant, τ₀ is the life time of the fluorescence in absence of quencher and τ₀ of BSA ≈10⁻⁸ s,³⁷ [Q] is the concentration of the quencher, and K_SV is the Stern–Volmer quenching constant. Fig. 3 shows the Stern–Volmer plots for the BSA fluorescence quenching by procyanidin B3. The values of K_SV and k_q for the interaction of procyanidin B3 with BSA at four different temperatures are shown in Table 2. In the present case, a linear Stern–Volmer plot is observed for procyanidin B3, which means that only one type of quenching mechanism occurs (dynamic or static). Dynamic and static quenching can be distinguished by their different dependence on temperature. For dynamic quenching, higher temperatures result in faster diffusion and larger amounts of collisional quenching. The quenching constant increases with increasing temperature, but the reverse effect would be observed for static quenching.³⁸ The results show that the K_SV values decrease with increasing temperature, and the values of k_q are greater than the limiting diffusion rate constant of the biomolecule (2 × 10¹⁰ L mol⁻¹ s⁻¹)³⁹ (Table 2), which suggests that the quenching mechanism of BSA by procyanidin B3 is not initiated by dynamic quenching but by static quenching.³⁴

Fig. 3 Stern–Volmer plots of BSA fluorescence quenched by procyanidin B3 at four different temperatures and pH 7.40.

Table 2 The quenching constants (K_SV), bimolecular quenching constants (k_q), binding constants (K_a) and the number of binding sites (n) of the procyanidin B3–BSA system at different temperatures

T (K)	KSV (L mol^−1)	kq (L mol^−1 s^−1)	Ka (L mol^−1)	n
293	2.538 × 10^4	2.538 × 10^12	2.257 × 10^4	0.974
298	2.487 × 10^4	2.487 × 10^12	2.307 × 10^4	0.958
303	2.409 × 10^4	2.409 × 10^12	2.416 × 10^4	1.024
310	2.406 × 10^4	2.406 × 10^12	2.512 × 10^4	1.131

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3.2.3. Binding parameters. The binding parameters are helpful in the study of pharmacokinetics. When small molecules bind independently to a set of equivalent sites on a macromolecule and the equilibrium between the free and the bound molecules has been reached, the fluorescence intensities obey the following equation:⁴⁰F₀ and F are the fluorescence intensities before and after the addition of the quencher, respectively. K_a is the apparent binding constant to a set of sites, and n is the average number of binding sites per BSA. [Q] and [P] are the total quencher concentration and the total protein concentration, respectively. By the plot of log(F₀ − F)/F vs. log(1/([Q] − (F₀ − F)[P]/F₀)), the number of binding sites n and binding constant K_a can be obtained. The results for procyanidin B3 at four different temperatures are given in Fig. 4 and Table 2. Binding parameters calculating from eqn (5) show that procyanidin B3 binds to BSA with the binding affinities of the order 10⁴ L mol⁻¹ and the binding sites n approximately equal to 1. These results are consistent with the results of ITC studies.

Fig. 4 The plots of log(F₀ − F)/F vs. log(1/([Q] − (F₀ − F)[P]/F₀)) for procyanidin B3–BSA system at four different temperatures and pH 7.40.

3.2.4. The equilibrium fraction of unbound procyanidin B3. The equilibrium fraction of unbound procyanidin B3 f_u is an important pharmacokinetic parameter, which influences procyanidin B3 elimination and distribution in the body. Generally, the free procyanidin B3 is available for diffusion and transport across cell membranes to reach the target site.³⁹ The free procyanidin B3 concentration may play a key role in the determination of procyanidin B3 action. Leonid M. Berezhkovskiy⁴¹ obtained the eqn (6) which can be used to calculate the equilibrium fraction of unbound procyanidin B3.where K_d is the equilibrium dissociation constant and K_d = 1/K_a.

A plot of the equilibrium fraction of unbound procyanidin B3 f_u at different ratios is presented in Fig. 5. As can be seen from the Fig. 5, f_u > 90% at the investigated concentration range and increases with the increase of procyanidin B3 concentration. The result shows that the concentration of free procyanidin B3 in plasma is enough to be stored and transported from the circulatory system to reach its target site to provide their therapeutic effects.

Fig. 5 The equilibrium fraction of unbound procyanidin B3 f_u for different [procyanidin B3]/[BSA] ratios at 298 K and pH 7.40.

3.2.5. Site-selective binding of procyanidin B3 on BSA. The drug competition for binding sites on serum albumin can also affect the free and bound forms of procyanidin B3. Therefore, it is important to identify the binding site of procyanidin B3 in BSA. BSA is composed of three structurally homologous, predominantly helical domains (I, II, and III), each containing two subdomains (A and B), and the principal regions of drug-binding sites on albumin are located in hydrophobic cavities in subdomains IIA and IIIA referred to as Sudlow's site I and II respectively.¹¹ Warfarin, an anticoagulant drug, and ibuprofen, a non-steroidal anti-inflammatory agent, have been considered as stereotypical ligands for Sudlow's site I and II, respectively.⁴² To identify procyanidin B3 binding site on BSA, competitive binding experiment was carried out, using warfarin and ibuprofen as site markers. Then information of the binding site that procyanidin B3 binds to can be obtained by monitoring the changes of the fluorescence of BSA after binding procyanidin B3, in the presence of warfarin and ibuprofen. As shown in Fig. 6A and B, with the addition of site marker (warfarin or ibuprofen) into BSA, the fluorescence intensity is lower than that of without site marker. To facilitate the comparison of the influence of warfarin and ibuprofen on the binding of procyanidin B3 to BSA, the binding constant in the presence of site markers was analyzed using the eqn (5) (Fig. 6C and Table 3). The binding constant is surprisingly variable in the presence of warfarin, while a smaller influence in the presence of ibuprofen (somewhat lower than with isolated BSA). The result indicates that the binding site of procyanidin B3 is mainly located within site I of BSA.

Fig. 6 Effect of site marker to procyanidin B3–BSA system (A and B) at 298 K and pH 7.40; λ_ex = 280 nm. c(warfarin) = c(ibuprofen) = c(BSA) = 2 × 10⁻⁶ mol L⁻¹; c(procyanidin B3)/(10⁻⁵ mol L⁻¹) (a–j): 0; 0.2; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0. The insets correspond to the molecular structures of site marker warfarin (A) and ibuprofen (B). (C) The plots of log(F₀ − F)/F vs. log(1/([Q] − (F₀ − F)[P]/F₀)) of site marker competitive experiments of procyanidin B3–BSA system.

Table 3 Binding constants of competitive experiments for the interaction of procyanidin B3 with BSA at 298 K and pH 7.40

System	Site marker	Ka (L mol^−1)	n	R^a	S.D.^b
a R is the correlation coefficient.b S.D. is standard deviation.
Procyanidin B3–BSA	Blank	2.307 × 10^4	0.958	0.99198	0.05698
	Warfarin	1.924 × 10^4	0.453	0.97191	0.05369
	Ibuprofen	2.001 × 10^4	1.030	0.99322	0.05597

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3.2.6. Synchronous fluorescence. Synchronous fluorescence spectra can supply characteristic information about the molecular environment in the vicinity of fluorophore molecules, such as Trp or Tyr, and have several advantages, such as spectral simplification, reduction of the spectral bandwidth, and avoidance of different perturbing effects.⁴³ The spectrum is obtained through the simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them. When the wavelength intervals (Δλ) are stabilized at 15 nm or 60 nm, the synchronous fluorescence gives the characteristic information of Tyr or Trp, respectively.⁴⁴

The effects of procyanidin B3 on the synchronous fluorescence spectra of BSA are shown in Fig. 7. Fig. 7A shows small blue shift (from 341 to 339 nm) in the tryptophan emission maxima (Δλ = 60 nm). There is almost no shift in the maximum emission wavelength when Δλ = 15 nm at the investigated concentration range (Fig. 7B). This suggests that hydrophobicity surrounding Trp is slightly increased in the presence of procyanidin B3, while the microenvironment around the Tyr residues did not obviously change during the binding process.⁴⁴ In Fig. 7C, the curve of Δλ = 60 nm is lower than the curve of Δλ = 15 nm, which leads to the conclusion that Trp plays an important role during fluorescence quenching of BSA by procyanidin B3. This signified that procyanidin B3 approaches the Trp more than the Tyr.

Fig. 7 Synchronous fluorescence spectra of BSA in the presence of different concentrations of procyanidin B3 (Δλ = 60 nm (A) and Δλ = 15 nm (B)) at 298 K and pH 7.40. c(BSA) = 2.0 × 10⁻⁶ mol L⁻¹; c(procyanidin B3)/10⁻⁵ mol L⁻¹, (a–j): 0; 0.2; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0. (C) Quenching of BSA synchronous fluorescence by procyanidin B3.

3.3. Absorption spectroscopic studies

UV-vis absorption technique can be used to explore the structural changes of protein and to investigate protein–ligand complex formation.⁴² The UV-vis absorption spectra of BSA in the absence and presence of procyanidin B3 obtained by utilizing the mixture of procyanidin B3 and phosphate buffer at the same concentration as the reference solution are shown in Fig. 8. BSA has two absorption peaks, the strong absorption peak at about 213 nm reflects the framework conformation of the protein, the weak absorption peak at about 279 nm appears to be due to the aromatic amino acids (Trp, Tyr, and Phe).⁴⁵ With adding of procyanidin B3, the intensity peak of BSA at 213 nm decreases with a red shift and the intensity of the peak at 279 nm has minimal changes (Fig. 8). The results can be explained that the interaction between procyanidin B3 and BSA leads to the loosening and unfolding of the protein skeleton and increases the hydrophobicity of the microenvironment of BSA.⁴⁶

Fig. 8 UV-vis spectra of BSA in presence of different concentrations of procyanidin B3 at 298 K and pH 7.40. c(BSA) = 2 × 10⁻⁶ mol L⁻¹; c(procyanidin B3)/(10⁻⁵ mol L⁻¹) (a–d): 0; 0.2; 0.5; 1.0.

3.4. FT-IR spectroscopic studies

The FT-IR spectra can be used to directly analyze the effect of procyanidin B3 on secondary structures of BSA. FT-IR spectra of proteins exhibit a number of amide bands, which represent different vibrations of the peptide moiety. Among the amide bands of the protein, the amide I band (1700–1600 cm⁻¹, mainly C=O stretch) and amide II band (1600–1500 cm⁻¹, C–N stretch coupled with N–H bending mode) both have a relationship with the secondary structure of protein.⁴⁷ However, the amide I band is more sensitive to the change of protein secondary structure than the amide II band.⁴⁸ The FT-IR spectra of free BSA and the difference spectra after binding with procyanidin B3 in phosphate buffer solution were recorded (Fig. 9A). Since there is no major spectral shifting for the protein amide I band at 1653 cm⁻¹ (mainly C=O stretch) and amide II band at 1546 cm⁻¹ (C–N stretch coupled with N–H bending mode) upon interaction with the procyanidin B3, their intensities remarkably decreased upon interaction with procyanidin B3. It is important to note that the decrease in the intensity of the amide I band is due to the decrease of the proportion of protein α-helix structure, the result also suggests BSA conformational changes upon the procyanidin B3–BSA interaction.^49–54 Procyanidin B3 indeed exerts some influence on the polypeptide carbonyl hydrogen bonding network and finally the reduction of the protein α-helix structure, but the effect is rather weak. The chemical interaction between procyanidin B3 and BSA is not likely to be a covalent binding.^49,55

Fig. 9 (A) FT-IR spectra of free BSA and difference spectra [(procyanidin B3–BSA) − procyanidin B3] at pH 7.40; (B) Percentage of secondary structure motifs of the free BSA and its procyanidin B3 complexes at pH 7.40. c(BSA) = 1.0 × 10⁻⁴ mol L⁻¹; the molar ratio of procyanidin B3 to BSA is 1 : 10.

The infrared self-deconvolution with second derivative and curve-fitting procedures were used to determine the protein secondary structures in the presence of procyanidin B3. The component bands of the infrared amide I band are attributed as follows: 1615–1637 cm⁻¹ to β-sheet, 1638–1648 cm⁻¹ to random coil, 1649–1660 cm⁻¹ to α-helix, 1660–1680 cm⁻¹ to β-turn, and 1680–1692 cm⁻¹ to β-antiparallel structures, respectively.³⁸ A quantitative analysis of the protein secondary structure for the free BSA and its procyanidin B3 complexes have been carried out, and the results are shown in Fig. 9B and Table 4. Upon procyanidin B3 interaction, a decrease of α-helix from 50.58% (free BSA) to 48.12% (procyanidin B3–BSA) is observed. The reduction of protein α-helix content is accompanied by an increase in β-turn and random coil (Fig. 9B). The random coil increases from 11.67% (free BSA) to 14.20% (procyanidin B3–BSA) and the β-turn structure increases from 9.10% (free BSA) to 10.11% (procyanidin B3–BSA). The decrease in α-helix structure and the increase in random coil and β-turn suggest a partial protein unfolding.^56,57

Table 4 Secondary structure analysis for amide I region in free BSA and its procyanidin B3 complex at pH 7.40

Amide I (cm^−1) components	Free BSA	Procyanidin B3–BSA
1649–1660 cm^−1 α-helix	50.58%	48.12%
1615–1637 cm^−1 β-sheet	25.14%	25.58%
1638–1648 cm^−1 random coil	11.67%	14.20%
1660–1680 cm^−1 β-turn	9.10%	10.11%
1680–1692 cm^−1 β-antiparallel	3.51%	1.99%

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3.5. CD spectroscopic studies

To confirm the FT-IR results, the conformational changes of BSA after binding with procyanidin B3 were evaluated by CD spectra. CD is a sensitive technique to monitor the conformational changes in the protein. The CD spectra of BSA in the absence and presence of procyanidin B3 are shown in Fig. 10. As shown in Fig. 10, CD spectra of BSA exhibited two negative bands at 209 and 222 nm, which is characteristic of the typical α-helix structure of protein.⁵⁸ The binding of procyanidin B3 to BSA causes only a decrease in negative ellipticity at all wavelengths of the far-UV CD without any significant shift of the peaks, which clearly indicates the changes in the protein secondary structure, and a decrease of the α-helix content in protein.⁵⁹ The percentage of α-helix can be calculated using the following equation:⁶⁰where MRE₂₀₈ is the observed mean residue ellipticity (MRE) value at 208 nm, 4000 is the MRE of the β-form and random coil conformation cross at 208 nm, and 33 000 is the MRE value of the pure α-helix at 208 nm.where C_p is the molar concentration of the protein (BSA), n the number of amino acid residues (583 for BSA) and l is the path length (0.2 cm). The α-helix content of protein was calculated from eqn (7) and (8). It can be calculated that the native BSA solution has 55.95% of α-helix, while α-helix content of BSA decreases to 53.82%, 52.21% and 48.63% with the addition of procyanidin B3 in the mole concentration ratios of 1 : 10, 1 : 20, and 1 : 40, respectively. This result is in agreement with the result of the FT-IR experiment. The secondary structure contents are closely related to the biological activity of the protein and this means a loss of the biological activity of the proteins in blood plasma. The result suggests the occurrence of conformational change at the secondary structural level in the reaction between procyanidin B3 and BSA.⁶¹

Fig. 10 Circular dichroism spectra of BSA (2 × 10⁻⁶ mol L⁻¹) in the absence and presence of procyanidin B3. c(procyanidin B3)/10⁻⁵ mol L⁻¹, (a–d): 0; 2.0; 4.0; 8.0.

3.6. Molecular docking studies

Molecular docking of the ligand with macromolecules provides insight into the preferred binding location and can be exploited to corroborate experimental observations to a large extent. The most possible binding mode between procyanidin B3 and BSA is shown in Fig. 11, it is clear that the location of procyanidin B3 in BSA is in the warfarin binding site (site I, subdomain IIA). The amino acid residues lining this binding site are composed of Ala210, Phe211, Lys212, Ala215, His288, Ala291, Ala217, Arg218, Val343, Val344, Lys195, Lys199 and Trp212. Furthermore, docking of procyanidin B3 to BSA creates a hydrophobic environment near Trp212, which provides a good structural basis to explain the efficient fluorescence quenching of BSA in the presence of procyanidin B3. On the other hand, there are hydrogen interactions of atoms O and H of procyanidin B3 with the residues Lev219, Asp451, Sep287, Phe223 and Ala291 of BSA. The length of the hydrogen bond is, respectively, 1.90 Å, 2.05 Å, 2.30 Å, 2.73 Å and 2.04 Å. The results indicate that the formation of hydrogen bond decreases the hydrophilicity and increases the hydrophobicity to make the procyanidin B3–BSA system stable. These results are consistent with the binding mode studies and the competitive binding experiment as analyzed above.

Fig. 11 Docking results of procyanidin B3–BSA system. The residues of BSA are represented by solid lines and procyanidin B3 with ball and stick model for clarity. The hydrogen bonds between procyanidin B3 and BSA are represented using yellow broken line.

4. Conclusions

The binding mechanism of procyanidin B3 interacting with BSA was investigated using ITC as well as several spectroscopic techniques and molecular docking under simulated physiological conditions. Data from ITC experiments suggest that the binding of procyanidin B3 to BSA is driven by enthalpy and entropy, and the major driving forces are the electrostatic interaction and hydrophobic interaction. The obtained binding constant for procyanidin B3 with BSA is in the intermediate range so that it is not too low to prevent efficient distribution and is not so high to lead to decreased plasma concentration. The stoichiometric binding number n approximately equals to 1, suggesting that one molecule of procyanidin B3 combines with one molecule of BSA and no more procyanidin B3 binding to BSA occurs at concentration ranges used in this study. Fluorescence experiments suggest that procyanidin B3 can bind to BSA and quench the fluorescence of BSA. The quenching mechanism is of static type and due to the formation of a ground state complex. Procyanidin B3 binds to BSA with the binding affinities of the order 10⁴ L mol⁻¹ and the binding sites n approximately equal to 1. These results are consistent with the results of ITC studies. The equilibrium fraction of unbound procyanidin B3 f_u > 90% at the investigated concentration range shows that procyanidin B3 can be stored and transported from the circulatory system to reach its target site. Site marker competitive experiment and molecular docking demonstrate that procyanidin B3 is mainly located within site I of BSA. Additionally, as shown by the UV-vis absorption and synchronous fluorescence spectroscopy, the binding of procyanidin B3 to BSA has little effect on the microenvironment around Tyr residues but the effect is sufficient to perturb the environment in the vicinity of the Trp residue from polar to slightly nonpolar. The results of FT-IR and CD suggest that procyanidin B3 indeed exerts some influence on the polypeptide carbonyl hydrogen bonding network and finally the reduction of the protein α-helix structure.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21173071) and the Research Fund for the Doctoral Program of Higher Education of China (20114104110002).

Notes and references

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