Probing the binding interaction of human serum albumin with three bioactive constituents of Eriobotrta japonica leaves: Spectroscopic and molecular modeling approaches

Journal of Photochemistry and Photobiology B: Biology 148 (2015) 268–276

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Probing the binding interaction of human serum albumin with three bioactive constituents of Eriobotrta japonica leaves: Spectroscopic and molecular modeling approaches Qing Wang, Qiaomei Sun, Xiangling Ma, Zaisheng Rao, Hui Li ⇑ College of Chemical Engineering, Sichuan University, Chengdu 610065, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 24 September 2014 Accepted 27 April 2015 Available online 8 May 2015 Keywords: Human serum albumin Corosolic acid Maslinic acid Tormentic acid Molecular modeling

a b s t r a c t Corosolic acid (CRA), maslinic acid (MA), and tormentic acid (TA) are three kind of bioactive constituents of Eriobotrta japonica leaves. In this study, plasma protein binding model prediction suggested that the binding ability to HSA was CRA > MA > TA. Furthermore, fluorescence spectroscopy confirmed this prediction. The results from emission and time resolved fluorescence studies revealed that the emission quenching of HSA with CRA, MA, and TA were all initiated by static quenching mechanism. From molecular docking results and site marker competitive experimental results it was possible to make good estimates about CRA, MA, and TA mainly bound to subdomain IIA of HSA. 3D fluorescence, FT-IR and CD spectra indicated that the local conformation of HSA molecules was affected by the presence of CRA, MA, and TA, but at different extents. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Eriobotrya japonica Lindl is a rosaceous evergreen tree distributed in southeastern China and Taiwan. Astringent leaves of loquat have been used as nutrition supplements for chronic bronchitis, coughs, phlegm, high fever and gastroenteric disorders [1]. Corosolic acid (CRA), maslinic acid (MA), and tormentic acid (TA) are three kind of bioactive constituents of the leaf (Fig. 1). Some common fruits also contain these constituents. CRA was also found in A. valvataDunn [2]. MA and TA were also found in olive fruit [3]. Some researchers have revealed that CRA, MA, and TA exhibit anti-diabetes, antioxidant activities and anti-inflammatory while being devoid of prominent toxicity [4–6]. Since they offer a wide variety of biological activities, the importance of these bioactive compounds has in recent years resulted in widespread interests. Human serum albumin (HSA) is a principal extracellular protein corresponding to a concentration of 40 mg/mL in blood plasma [7]. Because of its properties of binding various ligands such as warfarin, lysolecithin, fatty acids, triterpenes and several dyes, HSA has been used as a model protein for diverse physicochemical and biophysical studies [8,9]. The interaction between drug and protein can affect the biological activity and toxicity of drugs in pharmacology [10,11]. Hence, the interaction of CRA/MA/TA with

⇑ Corresponding author. Tel.: +86 028 85405149. E-mail address: [email protected] (H. Li). http://dx.doi.org/10.1016/j.jphotobiol.2015.04.030 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

HSA should be considered for its relevance to transport, biological activity, and clearance [12]. In the present study, CRA, MA and TA were isolated from Eriobotrta japonica leaves. Then molecular modeling and multi-spectroscopic methods were used to examine the interactions of CRA, MA and TA with HSA and to show the differences between CRA–HSA, MA-HSA and TA–HSA interactions. A plasma protein binding (PPB) model, which describes the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of a molecule within an organism, implemented by Discovery Studio software 3.1, was used to predict the binding of CRA/MA/TA to HSA. Molecular docking was performed to reveal the binding of CRA/MA/TA to HSA using Discovery Studio software 3.1 (DS 3.1). Then the drugs-HSA interactions were studied using the fluorescence method. In addition, the effects of CRA, MA and TA on the local conformation of HSA molecule were also examined using 3D fluorescence spectroscopy, FT-IR, and CD spectroscopy.

2. Materials and methods 2.1. Reagents HSA (fatty acid free) was purchased from Sigma Aldrich and used directly. A 2.0  105 mol L1 stock solution of HSA was prepared in 0.1 mol L1 Tris-HCl buffer solution (pH = 7.40) containing 0.05 mol L1 NaCl.

Q. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 268–276

Fig. 1. Molecular structures of corosolic acid (CRA), maslinic acid (MA), and tormentic acid (TA).

E. japonica leaves were purchased from Hehuachi Chinese Herbal Medicinal Market (Chengdu, China). CRA, MA and TA were isolated and their structures were identified by comparison of the physical and spectral data with the reported research. Stock solutions of CRA, MA and TA (2.0  103 mol L1) were prepared by dissolution of the drugs in anhydrous ethanol. All solutions in this work were diluted to the required volume with Tris-HCl buffer solution prepared from triple-distilled water, and all the other reagents were of analytical grade. All stock solutions were stored at 0–4 °C.

2.2. Apparatus and methods ADMET and the CDOCKER docking program, implemented in DS 3.1 (State Key Laboratory of Biotherapy, Sichuan University, China), were used in the present study. For ligand preparation, the 3D structure of CRA, TA and MA were generated with ChemBioOffice 2010 and optimized with DS 3.1 and subjected to CHARMm force field simulation before docking [13]. The crystal structures of HSA (PDB ID: 2BXD and 2BXG) were obtained from Protein Data Bank for docking simulations. Chain A was deleted from 2BXD and 2BXG. In the protein structure, missing bond orders, hybridization states, charges, and angles were assigned, and explicit hydrogen was added with a pH of 7.40 [14]. The protein structure energy was minimized by using 200 steps of the smart minimize method. Fluorescence measurements at different temperatures (298, 310, and 318 K) were performed on a Cary Eclipse fluorophotometer (Varian, USA) equipped with 1.0 cm quartz cells. Steady-state fluorescence spectra were recorded at wavelengths ranging from 300 nm to 500 nm with an excitation wavelength of 280 nm. The slit widths for excitation and emission were set to 5 nm. During the measurements, the protein concentration was 2.0  106 mol L1, and ligand concentrations of 1.0  105 mol L1 to 6.0  105 mol L1 were used for the measurements. Site marker competitive experiments were conducted by keeping the ratio of [HSA]/[drugs] = 1:2. Warfarin and ibuprofen were then gradually added to the binary mixture of CRA/MA/TA-HSA. An excitation wavelength of 280 nm was selected, and the fluorescence spectra were recorded in the range of 300 nm to 500 nm. The 3D fluorescence spectra of HSA (2.0  106 mol L1) and the HSA–ligand

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complexes (ratio of [HSA]/[drugs] = 1:1) were obtained under excitation wavelengths ranging from 200 nm to 400 nm at 10 nm increments. The emission spectra were monitored from 200 nm to 500 nm. Fluorescence lifetime measurements were executed using a Horiba Jobin Yvon FluoroMax-4 spectrofluorometer (HORIBA, FRA). The time-resolved fluorescence quenching of HSA by the drugs were recorded by fixing 280 nm as the excitation wavelength whereas 345 nm was the emission wavelength. The HSA concentration was fixed at 2.0  106 mol L1 and CRA, MA, and TA concentration were varied from 2.0  105 mol L1 to 6.0  105 mol L1 at room temperature. The absorption spectra were performed using a TU-1901 UV–vis spectrophotometer (Persee, Beijing, China) at 298 K at a wavelength of 190 nm to 550 nm and a scan slit of 1 nm. The IR spectra were recorded on a Nicolet-6700 FT-IR (Thermo, USA) spectrometer with a smart OMNI-sampler accessory. The FT-IR spectra of HSA (2.0  104 mol L1) in the absence and presence of drugs (2.0  104 mol L1) were measured in the 4000– 600 cm1 range with a resolution of 4 cm1 and 64 scans at room temperature. The corresponding absorbance contributions of the buffer and free CRA/MA/TA solutions were recorded and digitally subtracted from the same instrumental parameters. The CD spectra were recorded on a CD spectrometer (Model 400, AVIV, USA). CD measurements were recorded by keeping the concentration of HSA constant (2.0  106 mol L1) while varying the complex concentration from 0 to 8.0  104 mol L1 (ri = [drugs]/[HSA] = 0:1, 4:1). Spectra were recorded at 298 K in a 2 mm pathlength quartz cell from 260 to 200 with a step size of 1 nm, band width of 1 nm, and an averaging time of 0.5 s.

3. Results and discussion 3.1. Isolation of triterpene acids from E. japonica leaves The dried and powdered leaves of E. japonica (1 Kg) extracted with MeOH (24 h 3  6 L) at room temperature. After evaporation of the solvent, the residue was suspended in H2O and EtOAc, and the org. phase was evaporated to dryness in vacuo to provide the EtOAc-soluble extract (18.3 g). The EtOAc part (18.3 g) was subjected to CC (SiO2, CHCl2/MeOH): Fractions 1–5. Fr. 5 (3.5 g) was submitted to CC (MCI, Me2CO/H2O): Fractions 5.1–5.4. Fr. 5.3 was purified by repeated CC (RP-C18, MeOH/H2O 90:10): CRA156 mg, MA50 mg, and TA57 mg, resp. All compounds were identified by their physical data (NMR, MS, IR and HPLC) and by comparison with published values [15].

3.2. Plasma protein binding (PPB) model prediction The PPB model predicts whether a compound is likely to be highly bound to carrier proteins in the blood. PPB makes this decision based on AlogP98 and 1D similarities between two sets of ‘‘marker’’ molecules. One set of markers is used for flag binding at a level of 90% or greater and the other set is used for flag binding at a level of 95% or greater. Each marker molecule has a characteristic 1D similarity threshold that is used to determine whether a given compound is sufficiently similar that it binds at the associated level of the marker (90% or 95%). Therefore, if a compound exhibits 1D similarity to any 90% marker molecule that meets or exceeds the marker threshold, the compound is flagged as likely to bind at 90% or higher. Binding at 95% or greater is predicted if the similarity threshold is exceeded for any marker in the 95% set. Binding level predictions were also supplemented by conditions on AlogP98 (Atom-based LogP from FastDesc) [16].

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Table 1 Key to ADMET–PPB level. Level

Description

0 1 2

Binding is <90% (no markers flagged and AlogP98 < 4.0) Binding is >90% (flagged at 90% or AlogP98 > 4.0) Binding is >95% (flagged at 95% or AlogP98 > 5.0)

The key to determining the ADMET–PPB level is shown in Table 1. The results indicated that the PPB level of TA is 1, whereas that of CRA and MA is 2. In addition, the AlogP98 of CRA is 5.53 and MA is 5.48 (Table 2). Based on Table 1 data and combined results, we concluded that CRA and MA were more likely to be highly bound to carrier proteins in the blood than TA. In addition, CRA was look easer bound to carrier proteins than MA because of the higher AlogP98. HSA is the major soluble protein in the circulatory system, which has many physiological functions, such as maintaining the osmotic pressure and pH of blood and transporting a wide variety of endogenous and exogenous compounds including fatty acids, steroids, metal, amino acids, and drugs. Therefore, we speculated that CRA, MA, and TA could bound with HSA and the binding ability was CRA > MA > TA. 3.3. Molecular modeling of the interaction of CRA, MA, and TA with HSA The HSA crystallographic analyses revealed that the protein contains three homologous a-helical domains (I to III). Domain I contained residues 1–195, domain II contained residues 196–383, and domain III contained residues 384 to 585. Each a-helical domain contained two subdomains (A and B). On the basis of the arguments above, molecular docking was able to determine the mainly binding sites in HSA. Molecular modeling, as a complementary application, has recently been widely employed to improve the understanding of drug interactions with HSA. Autodock and Sybyl have been commonly used in numerous studies on drug– protein interactions [17,18]. The CDOCKER docking program (a molecular dynamics (MD) simulated-annealing-based algorithm), implemented in Discovery Studio, is also a kind of effective means used for drugs interact with macromolecules [19–21]. The name of this program came from metallurgical annealing, which is a procedure involving the heating and controlled cooling of a material to improve the crystal orders and reduce their defects [22,23]. CDOCKER is a grid-based molecular docking method that employs CHARMm. For pre-docked ligands, prior knowledge of the binding site is not required. It is possible, however, to specify the ligand placement in the active site using a binding site sphere. Crystallographic analysis shows that HSA contain multiple binding sites where can bind a wide variety of ligands. Two primary sites (1 and 2), located in subdomains IIA and IIIA, are highly adaptable bind many commonly used drugs (e.g. warfarin,

Table 2 The PPB_Level, AlogP98, and Stern–Volmer quenching constants of the CRA/MA/TAB– HSA system.

a

System

PPB_Level

AlogP98

CRA–HSA

2

5.53

MA–HSA

2

5.48

TA–HSA

1

4.38

Is the correlation coefficient.

T/K

KSV/(104 M1)

Ra

298 310 318 298 310 318 298 310 318

1.32 1.21 1.06 0.56 0.45 0.41 0.16 0.14 0.13

0.9986 0.9979 0.9967 0.9952 0.9989 0.9953 0.9936 0.9949 0.9977

ibuprofen) [24]. The residues of sites 1 and 2 on the protein are key determinants of binding specificity [25]. In the present manuscript, 2BXD/2BXG was defined as the total receptor, and the site sphere was built with a diameter of 5 Å based on warfarin/ibuprofen. Pre-existing warfarin/ibuprofen was then removed. In 2BXD, warfarin clustered in the center of subdomain IIA pocket. In 2BXG, ibuprofen clustered in the center of the binding pocket of subdomain IIIA and oriented with at least one oxygen atom in the vicinity of the polar patch. However, ibuprofen also occupies a previously undetected secondary site at the interface between subdomains IIA and IIB in 2BXG. During docking, a freshly prepared CRA/MA/TA was added. CHARMm was selected as force field. Heating steps were set at 2000 K with heating target temperature of 700 K. Cooling steps were set at 5000 K with a cooling target temperature of 300 K. Afterward, 10 molecular docking poses saved for each site were ranked according to CDOCKER energy (ECD, kcal/mol). This score included internal ligand strain energy and receptor-ligand interaction energy, and was used to sort the poses of each input ligand. The pose with the highest –ECD was selected as the most suitable pose for subsequent analysis. The results show that CRA, MA, and TA have poses in subdomains IIA while have 0 poses in subdomain IIIA and the undetected site. CRA, MA, and TA mainly bound to subdomain IIA of HSA. The 2D docking of HSA with CRA, MA, and TA is presented in Fig. 2a–c. CRA, MA, and TA clustered in the centre of the subdomains IIA. Their space postures were very similar to warfarin (Fig. 3). The ligands invariably had a planar group pinned snugly between the apolar side-chains of L238 and A291. However, there was much greater variation in the drug position within the plane perpendicular to the line between these two residues. This was particularly evident at the mouth of the pocket where the wide opening and presence of flexible side-chains provides significant room for manoeuvre. This finding provided a structural basis for the efficient fluorescence quenching of HSA emission in the presence of CRA, MA, and TA. Fig. 2a and c shows that the Arg-257 and Ala-261 residues of HSA were capable of forming intermolecular H-bonds with hydroxyl of the A ring. In addition, Lys-199 and Arg-222 also formed H-bonds with carboxyl between A and C ring. Fig. 2b shows that the Arg-257 residue of HSA formed H-bonds with two hydroxyls of the A ring. Lys-199 of HSA formed only one H-bond with carboxyl between A and C ring. Therefore, the interaction between CRA/MA/TA and HSA was dominated by hydrogen bonds, but other forces may be present. The formation of hydrogen bonds decreased hydrophilicity and increased hydrophobicity, which stabilized the HSA–CRA/MA/TA systems. Spectral experiments were performed based on the findings of the software simulation to determine the binding mechanisms and validate the results. 3.4. Analysis of fluorescence quenching of HSA by CRA, MA and TA The fluorescence intensity decreased on a different level when the concentration of HSA was fixed at 2.0  106 M at room temperature after the addition of CRA, MA, and TA, respectively, as shown in Fig. 4. It is obvious that the order of the influence of pentacyclic triterpenes was determined as following: CRA > MA > TA. This finding is consistent with the PPB model prediction results. Compared with the structure of CRA, MA, and TA, the size and steric hindrance is one of the crucial factors to affect the interaction between drugs and HSA. Fluorescence quenching may result from a variety of processes such as static quenching, dynamic quenching, and a combination of these [26]. Considering dynamic quenching depends on diffusion and high temperatures to achieve larger diffusion coefficients, we expected the bimolecular quenching constants to increase with increasing temperature. Increased temperatures are likely to decrease the stability of complexes in static quenching systems,

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Fig. 2. 2D docking representation of the interactions of HSA with CRA (a), MA (b) and TA (c). Only important interacting residues are shown and represented as sticks. The hit compound is shown in pink. Residues involved in hydrogen bonding, charge, and polar interactions are represented by magenta circles. Residues involved in van der Waals interactions are represented by green circles. The solvent-accessible surfaces of atoms are represented by a blue halo around the atom. The diameter of the circle is proportional to the solvent accessible surface. Hydrogen bonding interactions with amino acid side chains are represented by a blue dash with the arrows directed toward the electron donor. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

thereby lowering the rates of constants. The fluorescence quenching data at different temperatures (298, 310, and 318 K) were analyzed using the Stern–Volmer equation (1) to determine the quenching mechanism:

F 0 =F ¼ 1 þ K sv ½Q 

ð1Þ

where F0 and F signify the steady-state fluorescence intensities with and without a quencher, respectively; Ksv is the Stern–Volmer

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Fig. 3. Superimposition of docked pose (line) and crystal bound pose (stick) in the subdomains IIA of HSA.

Fig. 4. Fluorescence spectra of HSA in the presence of CRA (a), MA (inset, b), and TA (inset, c): (A) CHSA = 2.0  106 mol L1, (B-G) CCRA/MA/TA = 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0  105 mol L1.

quenching constant; [Q] is the quencher concentration. Table 2 shows the Ksv of CRA, MA, and TA. The presence of CRA decreased the fluorescence of HSA relatively stronger and the presence of TA had little effect on the fluorescence of HSA. The results suggested that the different structures of CRA, MA, and TA quenched of HSA fluorescence differently. The results were consistent with the PPB prediction. Dynamic and static quenching can be distinguished by temperature dependence of the quenching: Higher temperatures result in faster diffusion and, consequently, larger amounts of collisional quenching. However, higher temperature will typically result in the dissociation of weakly bound complexes, and hence, lower static quenching [27]. Quenching may followed a static mechanism and was due to complexation. Time-resolved fluorescence spectroscopy is a tool that can probe the interaction between ligands and proteins. Using the fluorescence lifetime measurements can distinguish static and dynamic quenching. Formation of static ground-state complexes

do not decrease the decay time of the uncomplexed fluorophores. Dynamic quenching is a rate process acting on the entire excitedstate population, and thus decreases the mean decay time of the entire excited-state population. Time resolved fluorescence lifetime measurement was carried out to substantiate the static quenching mechanism between HSA and CRA/MA/TA. The data were analyzed by tail fitting method. The qualities of the fits were assessed by v2 values and residuals. Mean (average) fluorescence lifetimes (‹s›) for biexponential iterative fitting were calculated from the decay times and the pre-exponential factors (a) using the following relation [28]:

hsi ¼ a1 s1 þ a2 s2

ð2Þ

We chose the mean fluorescence lifetime as an important parameter for exploring the behavior of HSA molecular bound to CRA, MA, and TA. Table 3 exhibited a tiny decrease in the average lifetime of HSA with the concentration of CRA/MA/TA increasing.

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Q. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 268–276 Table 3 Lifetimes of fluorescence decay of HSA in Tris-HCl of pH 7.4 at different concentrations of CRA/MA/TA. System

c(drug) (105 mol L1)

s1

s2

a1

a2

‹s›

v2

Free HSA CRAHSA

0.0 2.0 4.0 6.0 2.0 4.0 6.0 2.0 4.0 6.0

3.421 2.073 1.678 1.312 1.756 1.472 1.211 1.879 1.713 1.462

6.773 6.592 6.501 6.351 6.489 6.367 6.434 6.491 6.356 6.333

0.091 0.048 0.046 0.045 0.048 0.044 0.057 0.049 0.049 0.054

0.909 0.952 0.954 0.955 0.952 0.956 0.943 0.951 0.951 0.946

6.468 6.375 6.279 6.124 6.262 6.152 6.136 6.265 6.128 6.070

1.106 1.031 1.141 1.070 1.186 1.066 1.024 1.158 1.118 1.061

MAHSA

TAHSA

The lifetime data showed that the average lifetime of the fluorophore of HSA decreased marginally from 6.468 ns to 6.124 ns (CRA)/6.136 ns(MA)/6.070 ns(TA) with increasing drugs. The bound CRA/MA/TA may directly influence the lifetime of fluorophore. The observed fluorescence was from the uncomplexed fluorophores [27]. Such a slightly change of the average fluorescence lifetime in all tested systems, suggested the formation of complexes between the drugs and HSA. These results were consistent with the fluorescence quenching analysis results. In addition, FRET is an electrodynamic phenomenon that can be explained using classical physics. The donor molecules typically emit at shorter wavelengths that overlap with the absorption spectrum of the acceptor [27]. However, absorption spectra of CRA, MA, and TA had very little overlap with fluorescence spectra of HSA (Fig. 5). A consolation could be drawn: FRET was almost impossible to be a mechanism of quenching. The fluorescence quenching was essentially a static mechanism due to the ground state complex formation. Fig. 6. Effect of site marker probe (ibuprofen and warfarin) on the fluorescence of CRA(j)/MA(N)/TA(d)-HSA.

3.5. Site marker competitive experiments Based on the method presented by Sudlow et al. [29], the following equation was used to determine the percentage (I) of displacement of a probe:

I ¼ F=F 0

case of ibuprofen. These results suggest that CRA/MA/TA mainly bound to Sudlow site 1 in HSA subdomain IIA [17]. This result is consistent with the information from molecular modeling.

ð3Þ

where F and F0 represent the fluorescence intensity of HSA– CRA/MA/TA in the absence and presence of the probe, respectively. Fig. 6 shows that increasing warfarin concentration significantly altered the fluorescence intensity of HSA–CRA/MA/TA system. By contrast, fluorescence intensity did not significantly change in the

3.6. 3D fluorescence studies 3D fluorescence spectroscopy can provide detailed information on the conformational and microenvironmental changes of proteins that combine with small molecules [30]. The 3-D fluorescence spectral characteristics of the two systems of interest are listed in Table 4 in terms of peak position (kex/kem) and intensity [31,32]. The decrease in the fluorescence intensity of the two peaks indicated that CRA/MA/TA had a combination effect with HSA. Peak I (kex = 225.0 nm and kem = 344.0 nm) mainly exhibited the fluorescence characteristics of polypeptide backbone structures. This peak was correlated with the secondary protein structure. The fluorescence intensity of peak I decreased after the addition of CRA/MA/TA, which indicated that the peptide strand structure of HSA changed. Peak II (kex = 280.0 nm and kem = 344.0 nm) mainly revealed the spectral behavior of Trp and Tyr residues. The insertion of CRA/MA/TA molecule into the surrounding microenvironment of the Trp residue caused local conformational changes that directly altered HSA conformation. 3.7. FT-IR spectroscopy

Fig. 5. Spectral overlaps of the absorption spectra of corosolic acid (CRA), maslinic acid (MA), and tormentic acid (TA) with the fluorescence spectra of HSA. CCRA = CMA = CTA = 1  105 mol L1. CHSA = 2  106 mol L1.

Recently, FT-IR has emerged as an efficient tool for the characterization of drug–protein interaction [33,34]. Most investigations have concentrated on Amide I band because it is sensitive to the change of protein secondary structure. The component bands of

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Table 4 3D fluorescence spectral parameters of HSA alone and in the presence of CRA, MA, and TA. System

Peak no.

Peak position (kex/kem (nm/nm))

Intensity

Free HSA

I II I II I II I II

225/346 280/344 225/343 280/344 225/344 280/343 225/345 280/343

382.82 378.20 311.91 342.42 343.03 315.01 358.61 362.72

CRAHSA MAHSA TAHSA

Fig. 7. FT-IR spectra and difference spectra of HSA in the region of 1800–1400 cm1.

amide I are distributed according to the well-established assignment criterion. The amide I peak occurs in the 1,600–1,700 cm1 region (mainly the C@O stretch) and is widely used for investigating the secondary structures of protein. In general, the band centered at approximately 1610–1640 cm1 corresponds to the b-sheets, that at approximately 1640–1650 cm1 corresponds to random coils, that at approximately 1650–1658 cm1 corresponds to the a-helix, and that at approximately 1660–1700 cm1 corresponds to the b-turns. A quantitative analysis of the protein secondary structure of HSA before and after the interaction with CRA/MA/TA in Tris-HCl buffer of pH 7.40 is shown in Fig. 7. The peak position of amide I and II was shift. These shifts were attributed to CRA/MA/TA binding to C@O, CAN and NAH groups (hydrophilic interactions) in the protein polypeptides. Based on the curve-fitting results for amides I (Fig. 8), the secondary structures of HSA were estimated in the absence and presence of CRA, MA, and TA. Free HSA consisted of 52% a-helix structures, 11% b-sheet structures, 18% random coils structures and 19% b-turn structures. However, X-ray structural analysis showed higher a-helix content for HSA (67%) than those obtained by spectroscopic methods. The reason can be due to the sample preparation and the protein structural arrangements in the solution (spectroscopy). But this does not affect estimate the change tendency of secondary structure. After addition of CRA to HSA, the content of a-helix decreased to 37%, whereas the contents of b-sheet structures, random coils and b-turn structures increased up to 12%, 19% and 32%, respectively. In addition, the content of a-helix structures in HSA decreased to 39%, whereas that of the random coils structures of changed to 15%; b-turn structures increased to 35% after binding with MA. The content of a-helix structures decreased to 40%, whereas the contents of b-sheet, random coils and b-turn structures increased up to 14%, 21% and 34% with the addition of TA. The observed spectral changes were due to

Fig. 8. The curve fitting of amide I of free HSA (a), CRA-HSA (b), MA-HSA (c), and TA-HSA (d).

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4. Conclusions

Fig. 9. The CD spectra of HSA in the absence and presence of drugs at pH 7.40. CCRA (b) = CMA (c, solid) = CTA (d, CHSA = 2.0  106 mol L1, dash) = 8.0  106 mol L1.

In this study, the interaction of CRA, MA, and TA with HSA were investigated by using molecular modeling and different optical techniques. CRA, MA, and TA were isolated from E. japonica leaves. The PPB modeling prediction provided a theoretical direction for obtaining more reliable experimental results. The fluorescence quenching mechanism of HSA by CRA, MA, and TA were consistent with a static model. The existence of static quenching mechanism was further confirmed by the time resolved fluorescence spectral analysis. The binding ability to HSA was CRA > MA > TA. This was due to differences in the molecular structures. From molecular docking results and site marker competitive experimental results it was possible to make good estimates about CRA, MA, and TA mainly bound to Sudlow site 1 (subdomain IIA) of HSA. The 3D fluorescence, FT-IR and CD spectra indicated that the local conformation of HSA molecules was affected by the presence of CRA, MA, and TA, but at different extents.

References the major perturbations. The drugs–HSA complexes caused the rearrangement of the polypeptide carbonyl hydrogen-bonding network [35]. CRA/MA/TA interacted with the C@O groups in the protein polypeptides. The electron density redistribution of amide function group decreased the intensity of the original vibrations [36]. CRA/MA/TA formed H-bonds with HSA. This finding is consistent with the molecular docking results. 3.8. Circular dichroism studies CD is a universally acknowledged technique used for the structural characterization of proteins [37]. CD spectroscopy enables the rapid determination of the protein secondary structure and rapid assessment of the conformational change caused by ligand addition. The CD spectra of HSA in the absence and presence of CRA, MA, and TA were shown in Fig. 9. As can be seen, HSA exhibited two negative ellipticities at 208 and 220 nm, which were characteristic of the a-helix structure of proteins. The shapes of CD spectra were similar with and without CRA/MA/TA, which suggests that the HSA structure was also predominantly a-helix. This finding indicated that CRA/MA/TA bound with the amino acid residues of the main polypeptide chain of the protein and disrupted the hydrogen bond networks. CD results were generally expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol1 according to the following equation [38,39]:

MRE ¼

observedCDðm degÞ 10C p nl

ð4Þ

where Cp is the molar concentration of the protein, n is the number of amino acid residues (585 for HSA), and l is the path-length of the cell (here 2 mm). The a-helix contents of free and combined HSA were calculated from the MRE values at 208 nm by using the following equation:

a-helixl ð%Þ ¼

MRE208  4000 33000  4000

ð5Þ

The calculated results exhibited a decrease in a-helix content from 54.17% in free HSA to 50.11%, 51.97%, and 52.09% in the CRA-HSA, MA-HSA, and TA-HSA complexes (molar ratio, 1:4), respectively. The fact that its a-helix content decreased indicated that the HSA secondary structure changed during its reaction with CRA/MA/TA. This result was also in agreement with the results of the IR experiment.

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