Interactions between forsythoside E and two cholinesterases at the different conditions: fluorescence sections

Forsythoside E with the purity of 98.6% was purchased from Must (Chengdu, China). AChE from electric eel (200 U/g, EC 3.1.1.7) and BChE from equine serum (4000 U/g, EC 3.1.1.8; P06276) were purchased from Macklin (Shanghai, China) and Aladdin (Shanghai, China), respectively. Amyloid-β 25–35 (Aβ_25–35) was purchased from Sangon Biotech (Shanghai, China). S-Acetylthiocholine iodide (AsCh) and S-Butyrylthiocholine iodide (BsCh) were purchased from Sangon Biotech (Shanghai, China) and Sigma-Aldrich (Shanghai, China), respectively. CuCl₂·2H₂O and MgCl₂·6H₂O were purchased from Beijing Red Star Chemical (Beijing, China), and FeCl₃·6H₂O was purchased from Tianjin Tianli Chemical (Tianjin, China). All other reagents used in the work are the analytical grade. The other stock solution was made in the purified water except the stock solution of AChE and BChE was made in PBS (pH of 7.4). Unless otherwise specified, the concentration of the compound in the solution was the final concentration.

The fluorescent spectra were recorded using the fluorescence spectrophotometer (Fluromax-4, HORIBA Scientific, USA) which was equipped with the quartz cuvette of 1 cm. The fluorescence was recorded five minutes after adding the chemical into the solution. The fluorescence of AChE and BChE were recorded between 290 and 500 nm with the excitation wavelength of 280 nm. And the excitation and emission slit width was set as 10 nm.

All the spectroscopic recordings were performed at room temperature (RT), and were repeated at least twice. All the spectroscopic recordings were conducted in the solution with pH of 7.4 except where designated. The concentrations of AChE and BChE both were kept at almost 1 μM in the same volume in the experiments of the fluorescent recordings.

The quenching rate constant (k_q) and the Stern–Volmer quenching constant (K_sv) between forsythoside E and BChE were calculated by using Stern–Volmer plots: F₀ versus F and the equations:

$$\begin{eqnarray}&&{F}_{0}/F=1+{k}_{{\rm{q}}}{\tau }_{0}[Q]=1+{K}_{{\rm{sv}}}[Q]\end{eqnarray} \tag{ 1 }$$

$$\begin{eqnarray}&&{{\rm{k}}}_{{\bf{q}}}=\displaystyle \frac{{{\rm{K}}}_{{\bf{s}}{\bf{v}}}}{{\tau }_{{\bf{0}}}}\end{eqnarray} \tag{ 2 }$$

where F₀ and F are the fluorescence intensities of BChE in the absence and presence of forsythoside E, respectively; τ₀ is the average integral fluorescence lifetime of BChE and [Q] is concentration of forsythoside E.

The binding constant (K) and the number of the binding sites (n) of forsythoside E binding with AChE were gotten from the modified Stern–Volmer plots: lg[(F − F₀)/F₀] versus lg[Q] and the equation:

$$\begin{eqnarray}&&\mathrm{lg}\,[(F-{F}_{0})/{F}_{0}]=\,\mathrm{lg}\,K+n\,\mathrm{lg}\,[Q]\end{eqnarray} \tag{ 3 }$$

where F₀ and F are the fluorescence intensities of AChE in the absence and presence of forsythoside E, respectively, [Q] is concentration of forsythoside E, K denotes the binding constant of forsythoside E with AChE and n is the number of binding sites.

The binding constant (K) and the number of the binding sites (n) of forsythoside E binding with BChE were gotten from the modified Stern–Volmer plots: lg[(F₀ − F)/F] versus lg[Q] and the equation:

$$\begin{eqnarray}&&\mathrm{lg}\,[({F}_{0}-F)/{F}_{0}]=\,\mathrm{lg}\,K+n\,\mathrm{lg}\,[Q]\end{eqnarray} \tag{ 4 }$$

where F₀ and F are the fluorescence intensities of BChE in the absence and presence of forsythoside E, respectively, [Q] is concentration of forsythoside E, K denotes the binding constant of forsythoside E with BChE and n is the number of binding sites.

FT-IR spectra were recorded using a Thermo Fisher Scientific Nicolet IS5 equipped with the germanium attenuated total reflection (Thermo Nicolet Co., USA) at RT, according to the previous method with slight modifications [25]. The recording range was 4000–400 cm⁻¹ at the resolution of 4 cm⁻¹, with 32 scans per spectrum.

After the crystal structure of AChE (with the code of 1EVE) and BChE (with the code of 5DYW) was downloaded from the RCSB PDB database, docking simulation for forsythoside E with AChE (or BChE) was carried out using Autodock 4.2.6 program based on the reported methods [26]. Based on the docking between the first compound and the protein, the docking between the protein and the second compound was studied.

The fluorescent spectroscopy is one of the valuable tools to study compound-protein interaction. The common feature of AChE and BChE is the distinct fluorescent emission at around 340 nm with the 280-nm exciting length [21]. Forsythoside E showed almost no intrinsic fluorescent emission at 340 nm with λ_ex of 280 nm. The effects of forsythoside E on the fluorescence of the two cholinesterases were studied by successive adding the compound into the cholinesterase solution. As shown in figure 2(a), the fluorescent intensity of AChE regularly increased as the concentration of forsythoside E increased at 15, 30, 45, 75, 105, and 135 μM. And, the fluorescence was blue shifted, that was, the fluorescent peak gradually moved towards the shorter wavelength, which may be due to forsythoside E binding to AChE resulting in a more hydrophobic amino acid micro-environment. BChE fluorescence decreased regularly with the addition of forsythoside E at 15, 30, 45...... μM, with the blue shift of the fluorescent peak (figure 2(b)).

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Effects of forsythoside E on the fluorescence of AChE and BChE in the presence and absence of Aβ, AsCh, or BsCh were studied by changing the order in which the compounds were added. Forsythoside E with the final concentration of 30 μM was first added to the AChE solution, and then Aβ was added in turn into the mixture. As shown in figure 3(a), when forsythoside E was added to the AChE solution, the AChE fluorescence increased significantly. The fluorescence gradually decreased with the increase of Aβ concentration of 1, 5 and 10 μM, with the reduction rates of 5.21%, 7.06% and 9.14%, respectively. Change the order in which forsythoside E and Aβ were added in the AChE solution. Firstly, Aβ at the different final concentration 1, 5 and 10 μM was added separately to the AChE solution, and then forsythoside E was added into the mixture with the final concentration of 30 μM. As seen in in figure 3(b), Aβ (1, 5 and 10 μM) slightly decreased the fluorescence of AChE in a concentration-dependent matter, while forsythoside E of 30 μM significantly enhanced it with the rising rate of 83.02%, 78.99% and 82.57%, respectively. In AD, the interactions between AChE with Aβ_12–28 yielded the complex AChE-Aβ which strongly stimulated rat Aβ aggregation, recruited endogenous Aβ, and caused neurotoxicity [27, 28]. Aβ_25–35 used in the study may not form complexes with AChE, so the structure of AChE was not affected. Even if Aβ_25–35 was added first, it did not affect the action of forsythoside E on AChE.

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Forsythoside E of 30 μM was first added to the AChE solution to strengthen fluorescence, and then AsCh at the final concentration of 1, 11, 21 and 31 μM was added in turn into the mixture, with the reduction rates of 0, 4.18%, 7.27% and 11.44% in figure 3(c), respectively. Change the order in which forsythoside E and AsCh were added in the AChE solution. As shown in figure 3(d), AsCh at the final concentration of 1, 11, 21 and 31 μM was, respectively, added to the AChE solution, the endogenous fluorescence of AChE was quenched in a concentration-dependent manner. Whereafter forsythoside E of 30 μM was added to the mixture solution, the AChE fluorescence increased significantly with the the rising rate of 69.33%, 66,79%, 72.01%, and 64.93%, respectively. In the study, the quaternary group of the choline moiety of AsCh was similar to that of ACh, so AsCh was used to act as ACh. The charged quaternary group of the choline moiety of AsCh interacted with the 'anionic' subsite in AChE [8], and did not affect Tyr amino acid residues of AChE sphere surface where was the binding site of the interaction between forsythoside E and AChE [21]. Therefore, the addition of forsythoside E after AsCh still greatly enhanced the fluorescence of AChE.

Forsythoside E (30 μM) was first added to the BChE solution to decrease fluorescence, and then Aβ at the final concentration of 1, 5, 10 and 20 μM was added in turn into the mixture, with almost no reduction in fluorescence, as seen in figure 4(a). Change the order in which forsythoside E and Aβ were added into the BChE solution. As shown in figure 4(b), when Aβ at the different concentration was, respectively, added to the BChE solution, there was almost no change in fluorescence intensity. Then, when forsythoside E of 30 μM was added to the mixture solution, the BChE fluorescence was continuously reduced faintly, with the similar reduction to that of forsythoside E alone. Unlike AChE, Aβ did not form a complex with BChE, and thus did not affect BChE amino acid microstructure, so forsythoside E decreased BChE fluorescence intensity.

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Forsythoside E was first added to the BChE solution to decrease fluorescence, and then BsCh at the different concentrations was added in turn into the mixture, respectively, with almost no decrease in fluorescence in figure 4(c). Change the order in which forsythoside E and BsCh were added into the BChE solution. As shown in figure 4(d), when BsCh at the final concentration of 1, 11, 21, and 31 μM was, respectively, added to the BChE solution, there was little change in BChE fluorescence. When forsythoside E of 30 μM was added to the mixture solution, the BChE fluorescence was slightly decreased with the almost same reducing rate. The probable cause was that the concentration of BsCh was not enough to interact with BChE, and therefore did not interfere with forsythoside E and BChE interaction.

Interactions between forsythoside E and two cholinesterases were studied further by FT-IR. The FT-IR spectra of AChE, BChE, FE, AsCh, and AsCh were shown in figure S1, and the FT-IR spectra of of the interaction were shown in figure 5. A broad and strong characteristic band caused by the N–H asymmetric and symmetric stretching vibrations and the amide I band at 1665.25 cm⁻¹ induced by C=O stretching vibration of the peptide bond in AChE [25] were observed at 3307.45 and 3265.81 cm⁻¹, which slightly shifted in the direction of low wavenumber with the addition of forsythoside E, and then moved more after successively addition of AsCh (figure 5(a1)). Changing the order in which forsythoside E and AsCh were added, the spectrum had an almost same shifting pattern, except that the band at 1630.87 cm⁻¹ moved back a little after successively addition of forsythoside E (figure 5(a2)). Similarly, only the band at 1637.44 cm⁻¹ in BChE slightly shifted towards low wavenumber with the addition of forsythoside E, and kept unchanged after successively addition of BsCh, while the other two bands at 3274.57 and 1523.48 cm⁻¹ remained in the process of administration of forsythoside E and BsCh (figure 5(b1)). When BsCh was first added and then forsythoside E was added, there were exactly the same changes of bands (figure 5(b2)). These FT-IR results were similar to those of fluorescence.

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A molecular docking study was conducted using Autodock 4.2.6 program to discover the rationale behind the effects of forsythoside E on AChE and BChE (figure 6). The docking scores of forsythoside E and AsCh on AChE were −6.23 and −5.02 kcal mol⁻¹, respectively; while the docking scores of forsythoside E and BsCh on BChE were −3.11 and −4.32 kcal mol⁻¹, respectively, with three kinds of primary interactions of hydrogen bond, π-π stacking, hydrophobic effect. Compared with the separate interaction between forsythoside E and AChE, the three interaction forces between them all changed with the addition of AsCh: The three hydrogen bonds (1 with Tyr 121 and 2 with Arg 289) were reduced to two (2 with Tyr 130), the one π-π stacking (1 with Phe 331) was increased to two (1 with Tyr 116 and 1 with Tyr 130), and hydrophobic action changed from (with Phe 330, Phe 331, and Tyr 334) to (with Trp 84, Gly 118, Gly 123, Ser 124, Leu 127, and Gly 441). Forsythoside E was offset at the docking point of AChE possibly because AsCh and forsythoside E were too close to the binding point of AChE, resulting in crowding out. After the addition of BsCh, the two interaction forces between forsythoside E and BChE were almost the same as their individual interaction: Four hydrogen bonds (1with Asn 228, 1with Asp 304, 1with Tyr 396, and 1with Pro 230) and hydrophobic action (with Pro 230 and Met 302), due to that the binding point of forsythoside E and BsCh is far different. These results may initially explain the results of fluorescence and infrared data.

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The different organs and tissues in living systems have different acid–base environments, so interactions between forsythoside E and cholinesterases at different pH values were investigated in the paper. As shown in figure S2, forsythoside E (15–135 μM) increased the fluorescence intensities of AChE and decreased the fluorescence intensities of BChE at pH of 6.4, 7.4 and 8.0. It was seen in figure 7(a) that the enhancement rate of forsythoside E to AChE fluorescence was the highest in acid solution (pH 6.4), the second in neutral solution (pH 7.4) and the lowest in alkaline solution (pH 8.0). But figure 7(b) showed that the reduction rate of forsythoside E to BChE fluorescence from high to low was alkaline solution (pH 8.0), neutral solution (pH 7.4) and acid solution (pH 6.4). AChE and BChE functioned in the different stages of AD. pH values at the different parts in brain may slightly vary at different stages of AD. Further research was needed.

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The effects of the metal ions (Cu²⁺, Fe³⁺, and Mg²⁺) on the cholinesterase fluorescence were studied by adding the metal ions into protein solution. As seen in figure S3, the metal ions at 0.5 μM slightly reduced the fluorescence intensities of AChE and BChE, suggesting that the metal ions may interact with AChE and BChE, and the combination with the former was stronger than with the latter.

Then the effects of the metal ions (Cu²⁺, Fe³⁺, and Mg²⁺) on the interaction between forsythoside E and cholinesterases were studied, and the fluorescen spectra were shown in figure S4. Metal ions increased the fluorescence enhancement rate of forsythoside E to AChE and the fluorescence quenching rate of forsythoside E to BChE. In the presence of Fe³⁺, the change was the highest, followed by Cu²⁺, and the lowest when Mg²⁺ were present, shown in figures 8(a) and (b).

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Two quenching modes of the protein fluorescence induced by a drug include the dynamic and the static mode. The quenching mode of BChE fluorescence induced by forsythoside E may be further characterized by the following method. Stern–Volmer plots and the corresponding equations (1) and (2) were used to calculate the quenching rate constant (k_q) and the Stern–Volmer quenching constant (K_sv) [29]. The modified Stern–Volmer plots: lg[(F−F₀)/F₀] or lg[(F₀−F)/F₀] versus lg[Forsythoside E] and the corresponding equations (3) and (4) were used to get the binding constant (K) and the number of the binding sites (n) of forsythoside E binding with cholinesterases [30]. The plots were showed in figures S5 and S6, and the results were summarized in table 1 and table 2.

The k_q values of BChE were higher than the maximum value of the quenching constant in the scatter collision (2 × 10¹⁰ M⁻¹s⁻¹) , which suggested that the quenching was not triggered by the dynamical collision but a complex formation between forsythoside E and BChE, and the static quenching was the main quenching mode in the process of forsythoside E binding with BChE [31]. The K values of ChE-forsythoside E were pretty much the same, implying that the interaction between ChEs and forsythoside E was almost unaffected by acid–base environment and metal ions. The n numbers of two ChEs approximately equaled to one, indicating that there was only one site on each cholinesterase applicable for forsythoside E to bind to. It may also be that metal ions and forsythoside E occupied the different active sites when ChEs were acted on. In the presence of metal ions, the binding constant K decreased slightly, indicating that the presence of metal ions weakened the interaction between forsythoside E and ChEs, that was, the ChEs could not be well inhibited. The metal ion hypothesis held that the disorder of metal ion metabolism may aggravate the process of AD, and the experimental results further indicated that the treatment of AD will be affected when the concentration of metal ions was not within the normal range. Moreover, the negative signs of ΔG for both cholinesterases demonstrated that there was spontaneous binding between forsythoside E and BChE [32].