# Insight into the Interaction between the HIV-1 Integrase Inhibitor Elvitegravir and Bovine Serum Albumin: A Spectroscopic Study

Authors: Ali Saber Abdelhameed
Journal: Journal of Spectroscopy
 (2015)

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## Abstract

The interaction between the anti-HIV drug Elvitegravir (EVG) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy and UV-visible absorption spectra. The mechanism for quenching the fluorescence of BSA by EVG is discussed. It was found that EVG can quench the intrinsic fluorescence of BSA through a static quenching procedure. The quenching type, association constant, and number of binding sites were investigated. The binding constant of EVG with BSA was calculated at different temperatures based on fluorescence quenching results. The thermodynamic parametersΔHθ, ΔGθ, and ΔSθ were determined. The positive ΔSθ and negative ΔHθ and ΔGθ values showed that a spontaneous interaction may involve both roles of hydrophobic interaction and hydrogen bonding. The interaction of BSA with EVG was also confirmed by UV absorption spectra. The average distance, r, between donor (BSA) and acceptor (EVG) was obtained according to Förster’s theory of nonradiation energy transfer. Synchronous fluorescence and three-dimensional fluorescence spectra were used to investigate the conformational change of BSA molecules that occur upon addition of EVG and showed, upon binding, a possibility of increasing hydrophobicity around tryptophan residues of BSA.


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## Body

## 1. Introduction

Serum albumins are the major soluble protein constituents of the circulatory systems, and they have many physiological functions [1, 2]. They act as storage and transport carriers for various endogenous and exogenous compounds [3, 4]. Therefore, interaction of drugs with and competition for the binding sites on albumins may strongly affect the distribution and elimination rate of the drugs as well as their action mechanism and pharmacodynamics. Hence, investigating the drug binding with albumins will facilitate interpretation of the metabolism and transport process of the drug and will help to elaborate the relationship between structures and functions of proteins. We have selected bovine serum albumin (BSA), for which association constants and binding sites are known for a number of compounds [5, 6]. BSA and human serum albumin (HSA) display approximately 76% sequence homology [7, 8]. Similar to other serum albumin, bovine serum albumin (BSA) is a multifunctional protein that possesses various physiological functions including binding, transport, and delivery of fatty acids [9, 10], porphyrins [11], bilirubin [12], and several other molecules. BSA is also responsible for certain binding aggregation and conformational dynamics in solution [13]. BSA has been extensively studied [14–16], and its molecular interactions with several molecules are often determined via spectroscopic techniques due to their high sensitivity, reliability, and ease of use [17–21]. On the other hand, integrase inhibitors are one of the more recently introduced classes of antiretroviral [22, 23]. Elvitegravir (EVG) (formerly GS-9137, JTK-303) (6-(3-Chloro-2-fluorobenzyl)-1-[(2S)-1-hydroxy-3-methyl-2-butanyl]-7-methoxy-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid) (Figure 1) is a potent, boosted, once daily, HIV integrase inhibitor through the inhibition of DNA strand transfer [24, 25]. EVG has antiviral activity against wild-type and drug resistant strains of HIV [24]. EVG inhibits the process of viral DNA strand transfer with the greatest potency 50% maximal inhibitory concentration [IC50] of <0.06 μmol·L−1 in vitro [26], thereby preventing HIV DNA from being integrated into host chromosomes and blocking provirus formation and infection propagation [27]. The inhibition of strand transfer occurs via the binding of Elvitegravir to the integrase-viral DNA complex and the drug’s subsequent interaction with the DNA and the essential magnesium ions within the active site of the integrase [22, 28]. EVG initially became available as part of a fixed-dose tablet (Stribild) in combination with emtricitabine, tenofovir disoproxil fumarate, and the pharmacokinetic boosting agent cobicistat in several countries, including North America and the EU [22]. Elvitegravir has also been approved in the EU and more recently (Sep. 2014) by the FDA as a single-agent tablet (Vitekta 85 or 150 mg) for use in combination with a ritonavir-boosted protease inhibitor plus one or more antiretrovirals for treatment-experienced HIV-positive adults [29, 30]. With the use of antiretroviral drugs, several potential interactions have to be considered. In the current study, the EVG induced quenching of the intrinsic fluorescence of BSA was investigated. BSA consists of a chain of 580 amino acid residues composing a well-defined single polypeptide sequence, which contains three homologous α-helix domains (I–III) [31, 32] and two tryptophan residues located at positions 134 and 212 of the chain [33]. The fluorescence intensity of BSA could be decreased by quenchers such as many drugs or small molecule organic compounds [34]. The changes of fluorescence or absorption spectra of BSA reflect the interaction of drugs and macromolecule protein. Extensive survey of the literature revealed that no reports studying EVG and serum albumin interactions were published. Hence, the current study is designed to study the interaction of EVG and BSA using fluorescence spectroscopy to explain the quenching type, the association constants, number of binding sites, and the basic thermodynamic parameters.Figure 1
Chemical structure of EVG.


## 2. Materials and Methods

### 2.1. Materials

Elvitegravir (EVG; CAS 697761-98-1) was purchased from MedChemexpress (NJ, USA). Bovine serum albumin (BSA) was purchased from Techno Pharmchem (Bahadurgarh, Haryana, India). HPLC grade methanol was purchased from BDH laboratory supplies (Poole, UK). Potassium dihydrogen orthophosphate anhydrous was purchased from Central drug house Ltd. (New Delhi, India). Sodium phosphate dibasic, anhydrous, was purchased from Bio Basic Inc. (NY USA). Extra pure sodium chloride purchased from Loba Chemie Pvt. Ltd. (Mumbai, India) Potassium chloride was purchased from WINLAB Ltd. (Leicestershire, UK). Ultrapure water of18μΩ obtained from a Millipore Milli-Q UF-Plus purification system (Millipore, Bedford, MA, USA) was used throughout the study.


### 2.2. Apparatus

A Jasco FP-8200 spectrofluorometer (JASCO International Co. Ltd. Tokyo, Japan) was used for all the fluorescence measurement, with excitation and emission slits at 5 nm,λex = 280 nm and 1-cm quartz cell. In the study, BSA was excited at 280 nm and monitored at 338 nm. All absorption spectral recordings and absorbance measurements were performed on a Cary 50 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). All measurements were performed in 1X phosphate buffered saline (PBS buffer) pH 7.4; the pH was measured on an Adwa AD1030 pH-meter (ADWA Instruments Inc., Romania).


### 2.3. Sample Preparation

EVG standard solution was prepared in methanol yielding a final concentration of 1.0 mg mL−1. A volume of 1.0 mL stock solution was diluted 10 mL with methanol to give a 100 μg mL−1 concentration as a working standard solution. The working solution was further diluted with PBS buffer pH 7.4 to produce a working standard solution of 20 μg mL−1.Bovine serum albumin (BSA) solution was prepared in a concentration of 1.0 mg mL−1 by dissolving 0.1 g pure BSA in PBS buffer pH 7.4 and completing the volume to 100 mL and kept in the cool, dark. This solution was further diluted with PBS buffer pH 7.4 to produce a working standard solution of 100 μg mL−1.


### 2.4. Procedures

#### 2.4.1. EVG-BSA Interactions

Upon optimizing the concentrations of both analytes, the BSA concentration was fixed at 50μg mL−1 with EVG concentration that ranged from 0.1 to 15 μg mL−1. Fluorescence measurements were performed at three temperatures (288, 298, and 309 K) in the range of 290–500 nm upon excitation at 280 nm. The three-dimensional fluorescence spectrum was performed using the initial excitation wavelength set at 240 nm up to 350 nm with an increment of 2 nm. Emission wavelength was set between 220 and 600 nm and an increment of 0.5 nm. Synchronous fluorescence was performed with a Δλ values of 15 and 60 scanning between 260 and 500 with a 0.5 nm data interval.


#### 2.4.2. UV Measurements

The UV-vis spectra of BSA were recorded for the drug-free protein and upon addition of EVG scanning in the wavelength range of 220–500 nm. BSA concentration was kept constant at 0.8 mg mL−1 while the drug concentration was 3.0, 5.0, 7.0, and 10.0 μg mL−1.


#### 2.4.3. Energy Transfer between EVG and BSA

A concentration of 10.0μg mL−1 of EVG was used for the absorption measurement in the range of 220–500 nm. Meanwhile, BSA (50 μg mL−1) emission spectrum was determined in the wavelength range of 280–500 nm. Hence, the energy transfer was computed in accordance to Förster’s nonradiative energy transfer theory (FRET) [35] utilizing the overlapped UV absorption spectrum of EVG with the fluorescence emission spectrum of BSA.


## 2.1. Materials

Elvitegravir (EVG; CAS 697761-98-1) was purchased from MedChemexpress (NJ, USA). Bovine serum albumin (BSA) was purchased from Techno Pharmchem (Bahadurgarh, Haryana, India). HPLC grade methanol was purchased from BDH laboratory supplies (Poole, UK). Potassium dihydrogen orthophosphate anhydrous was purchased from Central drug house Ltd. (New Delhi, India). Sodium phosphate dibasic, anhydrous, was purchased from Bio Basic Inc. (NY USA). Extra pure sodium chloride purchased from Loba Chemie Pvt. Ltd. (Mumbai, India) Potassium chloride was purchased from WINLAB Ltd. (Leicestershire, UK). Ultrapure water of18μΩ obtained from a Millipore Milli-Q UF-Plus purification system (Millipore, Bedford, MA, USA) was used throughout the study.


## 2.2. Apparatus

A Jasco FP-8200 spectrofluorometer (JASCO International Co. Ltd. Tokyo, Japan) was used for all the fluorescence measurement, with excitation and emission slits at 5 nm,λex = 280 nm and 1-cm quartz cell. In the study, BSA was excited at 280 nm and monitored at 338 nm. All absorption spectral recordings and absorbance measurements were performed on a Cary 50 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). All measurements were performed in 1X phosphate buffered saline (PBS buffer) pH 7.4; the pH was measured on an Adwa AD1030 pH-meter (ADWA Instruments Inc., Romania).


## 2.3. Sample Preparation

EVG standard solution was prepared in methanol yielding a final concentration of 1.0 mg mL−1. A volume of 1.0 mL stock solution was diluted 10 mL with methanol to give a 100 μg mL−1 concentration as a working standard solution. The working solution was further diluted with PBS buffer pH 7.4 to produce a working standard solution of 20 μg mL−1.Bovine serum albumin (BSA) solution was prepared in a concentration of 1.0 mg mL−1 by dissolving 0.1 g pure BSA in PBS buffer pH 7.4 and completing the volume to 100 mL and kept in the cool, dark. This solution was further diluted with PBS buffer pH 7.4 to produce a working standard solution of 100 μg mL−1.


## 2.4. Procedures

### 2.4.1. EVG-BSA Interactions

Upon optimizing the concentrations of both analytes, the BSA concentration was fixed at 50μg mL−1 with EVG concentration that ranged from 0.1 to 15 μg mL−1. Fluorescence measurements were performed at three temperatures (288, 298, and 309 K) in the range of 290–500 nm upon excitation at 280 nm. The three-dimensional fluorescence spectrum was performed using the initial excitation wavelength set at 240 nm up to 350 nm with an increment of 2 nm. Emission wavelength was set between 220 and 600 nm and an increment of 0.5 nm. Synchronous fluorescence was performed with a Δλ values of 15 and 60 scanning between 260 and 500 with a 0.5 nm data interval.


### 2.4.2. UV Measurements

The UV-vis spectra of BSA were recorded for the drug-free protein and upon addition of EVG scanning in the wavelength range of 220–500 nm. BSA concentration was kept constant at 0.8 mg mL−1 while the drug concentration was 3.0, 5.0, 7.0, and 10.0 μg mL−1.


### 2.4.3. Energy Transfer between EVG and BSA

A concentration of 10.0μg mL−1 of EVG was used for the absorption measurement in the range of 220–500 nm. Meanwhile, BSA (50 μg mL−1) emission spectrum was determined in the wavelength range of 280–500 nm. Hence, the energy transfer was computed in accordance to Förster’s nonradiative energy transfer theory (FRET) [35] utilizing the overlapped UV absorption spectrum of EVG with the fluorescence emission spectrum of BSA.


## 2.4.1. EVG-BSA Interactions

Upon optimizing the concentrations of both analytes, the BSA concentration was fixed at 50μg mL−1 with EVG concentration that ranged from 0.1 to 15 μg mL−1. Fluorescence measurements were performed at three temperatures (288, 298, and 309 K) in the range of 290–500 nm upon excitation at 280 nm. The three-dimensional fluorescence spectrum was performed using the initial excitation wavelength set at 240 nm up to 350 nm with an increment of 2 nm. Emission wavelength was set between 220 and 600 nm and an increment of 0.5 nm. Synchronous fluorescence was performed with a Δλ values of 15 and 60 scanning between 260 and 500 with a 0.5 nm data interval.


## 2.4.2. UV Measurements

The UV-vis spectra of BSA were recorded for the drug-free protein and upon addition of EVG scanning in the wavelength range of 220–500 nm. BSA concentration was kept constant at 0.8 mg mL−1 while the drug concentration was 3.0, 5.0, 7.0, and 10.0 μg mL−1.


## 2.4.3. Energy Transfer between EVG and BSA

A concentration of 10.0μg mL−1 of EVG was used for the absorption measurement in the range of 220–500 nm. Meanwhile, BSA (50 μg mL−1) emission spectrum was determined in the wavelength range of 280–500 nm. Hence, the energy transfer was computed in accordance to Förster’s nonradiative energy transfer theory (FRET) [35] utilizing the overlapped UV absorption spectrum of EVG with the fluorescence emission spectrum of BSA.


## 3. Results and Discussion

### 3.1. Fluorescence Quenching of BSA Induced by EVG

Spectroscopic techniques, particularly fluorescence spectroscopy, are able to provide enormous amount of information on the ligand protein binding, such as type, mechanism and strength of the binding, and so forth. It has been previously established that quenching of fluorescence intensity, that is, fluorescence intensity reduction of a certain fluorophore, can occur via several mechanisms including but not limited to formation of a nonfluorescent complex and energy transfer, and so forth [15, 36, 37]. The fluorescence spectra of BSA in presence of different amounts of EVG were measured in the range of 288–500 nm upon excitation at 280 nm. EVG led to a concentration dependent quenching of the intrinsic fluorescence of BSA (Figures 2 and 3) without changing the emission maximum but with a slight change in peak shape at higher EVG concentrations (>10 μg mL−1). These results indicated that an interaction between EVG and BSA is taking place [38].Figure 2
Fluorescence spectra of BSA (50μg·mL−1) in the absence (A) and in presence of EVG at different concentrations. EVG concentrations were 0.1 μg·mL−1 (B), 0.3 μg·mL−1 (C), 0.5 μg·mL−1 (D), 0.7 μg·mL−1 (E), 1.0 μg·mL−1 (F), 3.0 μg·mL−1 (G), 5 μg·mL−1 (H), 7.0 μg·mL−1 (I), 10 μg·mL−1(J), 12 μg·mL−1 (K), and 15 μg·mL−1 (L).Figure 3
The Stern-Volmer (a) and Lineweaver-Burk (b) curves at different temperatures.
(a)
(b)Furthermore, the fluorescence quenching data were analyzed by the Stern-Volmer equation (1) [39] and Lineweaver-Burk equation (2) [40]. Consider(1)F0F=1+KSVQ=1+Kqτ0Q(2)F0-F-1=F0-1+KLB-1F0-1Q-1,where F0 and F are the steady-state fluorescence intensities of BSA in the absence and presence of EVG, respectively, KSV the Stern-Volmer quenching constant and Q is the concentration of quencher (EVG). Kq is the quenching rate constant, τ0 is the average lifetime of the protein without the quencher and KLB is the static quenching constant. Within certain concentration, the curve of F0/F versus Q (Stern-Volmer curve) would be linear if the quenching type is single static or dynamic quenching [17]; similarly, the curve of F0-F-1 versus Q-1 (Lineweaver-Burk curve) would be linear [41] for static quenching. If the quenching type is combined quenching (both static and dynamic), the Stern-Volmer plot is an upward curvature [42]. One way to distinguish dynamic from static quenching is to examine the temperature effect on the interaction of the drug to BSA. The Stern-Volmer and Lineweaver-Burk curves of EVG-BSA at different temperatures are shown in Figure 5. It can be seen that both Stern-Volmer and Lineweaver-Burk curves were linear, which illustrated that the quenching type was probably a static quenching.Moreover, Table1 demonstrates the gradual reduction of KSV and KLB values upon temperature increase that supports the idea of a static type quenching of BSA fluorescence [42]. This hypothesis was also backed up by the Kq values (quenching rate constant), which was computed using the following equation (3)Kq=KSVτ0.Since the fluorescence lifetime of the biopolymer (τ0) can be taken as 10−8 s−1 [43], then the Kq values summarized in Table 1 were calculated for EVG-BSA system to be in the order of 1012 L Mol−1 s−1. Previous reports have shown that the maximum scatter collision quenching constant, Kq of various quenchers with the biopolymer is 2 × 1010 L M−1 s−1 [43]. Ultimately, the Kq values of protein quenching procedure initiated by EVG are higher than the Kq of the scattered procedure. This indicated that the quenching is not initiated by dynamic collision but from the formation of a complex [41].Table 1
Stern-Volmer and Lineweaver-Burk correlation parameters for BSA-EVG interaction.
Temperature (T) (K)
Stern-Volmer parameters*
Lineweaver-Burk parameters*
K
SV  ×  104 (L mol−1)
K
q  ×  1012 (L mol−1 s−1)
r
2
K
LB  ×  105 (L mol−1)
r
2
288
4.13 ± 0.24
4.13
0.9890
4.66 ± 0.15
0.9949
298
4.03 ± 0.27
4.03
0.9867
3.14 ± 0.14
0.9979
309
3.91 ± 0.26
3.91
0.9879
2.43 ± 0.14
0.9983
*Average of four determinations.


### 3.2. Binding Constants and the Number of Binding Sites

Based upon the probability of a static quenching process is taking place between BSA and EVG and if it is assumed that there are independent binding sites to a set of equivalent sites on the protein, the apparent binding constant(K) and the number of binding sites (n) can be determined from [42, 44](4)log⁡F0-FF=log⁡⁡K+nlog⁡⁡Q,where K is the binding constant, reflecting the reaction degree of BSA and EVG; n is the number of binding sites, specifying the number of EVG bound to a BSA molecule. Thus, a plot of Log(F0-F)/F versus log Q could be used to determine K and n. The K and n at different temperatures are summarized in Table 2, indicating good assumptions made for deriving (4). Table 2 also demonstrates a decrease in the binding constant with the increase in temperature, yielding a less stable EVG-BSA complex. Additionally, n values were found to be ~1, which in turn may infer the involvement of one independent class of binding sites on BSA for EVG.Table 2
Binding constantK and the number of binding sites n.
Temperature (T) (K)
K  ×  103 (L mol−1)
n
288
2.68
0.67 ± 0.03
298
1.99
0.72 ± 0.02
309
1.12
0.75 ± 0.03


### 3.3. Thermodynamics and Interaction Forces between EVG and BSA

As the binding constant is dependent on temperature; hence, a thermodynamic process was regarded responsible for the complex formation. Therefore, temperature dependent thermodynamic parameters were determined for further investigating the acting forces between EVG and BSA. Generally, the force between a small molecule and a biological macromolecule includes hydrophobic interaction, hydrogen bonding, van der Waals force, and electrostatic force. The thermodynamic parameters associated with the different types of interactions were previously reported [45]. In which, hydrophobic force may increase ΔHθ and ΔSθ of a system, while van der Waals power may decrease them with negative ΔHθ indicative for hydrogen bonding and electrostatic force usually makes ΔHθ~0 and ΔSθ>0 [46]. Thermodynamic parameters, enthalpy (ΔHθ), and entropy (ΔSθ) of EVG-BSA interaction are significant to confirm binding mode. Binding studies were performed at 288, 298, and 309 K, at which BSA does not undergo any structural degradation. The thermodynamic parameters were evaluated using the Van’t Hoff and Gibbs-Helmholtz [47, 48] equations:(5)log⁡⁡K=-ΔHθ2.303RT+ΔSθ2.303RΔGθ=ΔHθ-T·ΔSθ,where K and R are the binding constant and gas constant, respectively. ΔHθ, ΔGθ, and ΔSθ are enthalpy change, free energy change, and entropy change, respectively. Therefore, a plot of  log⁡K versus 1/T (Figure 4) enables the determination of ΔHθ, ΔGθ, and ΔSθ by (5). Thermodynamic parameters for EVG-BSA binding are summarized in Table 3 and demonstrate that hydrogen bonding and hydrophobic interactions play a role in the binding between EVG and BSA.Table 3
Thermodynamic parameters of BSA-ELV binding.
Temperature (T) (K)
Δ
G
θ (kJ mol−1)
Δ
H
θ (kJ mol−1)
Δ
S
θ (J mol−1 K−1)
288
−42.64
−30.83
41.02
298
−43.05
309
−43.50Figure 4
Van’t Hoff plot for the binding between EVG and BSA, variation of lnK as a function of 1/T.Figure 5
The overlap of the fluorescence spectrum of BSA (a) and the absorbance spectrum of EVG (b).


### 3.4. Energy Transfer between EVG and BSA

The aforementioned spectral studies have suggested that EVG forms a nonfluorescent complex with BSA. Three types of intrinsic fluorophores are known to be found in BSA sequence, namely, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe); however the intrinsic fluorescence of BSA is mainly due to tryptophan [49]. The efficiency of energy transfer can be used to determine the distance, r, between the drug and the tryptophan residues in the protein. The distance r is an average value between the bound EVG and the two tryptophan residues of BSA (Trp-135 and Trp-214) and can be determined using Förster’s resonance energy transfer (FRET) [35, 44]. Generally, energy transfer occurs whenever the emission spectrum of a fluorophore (donor) overlaps with the absorption spectrum of another molecule (acceptor) and the distance between them is less than 8 nm. The overlap of the UV absorption spectrum of EVG with the fluorescence emission spectra of BSA is shown in Figure 5. The efficiency of energy transfer, E, is calculated using (6)E=1-FF0=R06R06+r6,where F and F0 are the fluorescence intensities of BSA in the presence and absence of EVG, r is the distance between acceptor and donor and R0 is the critical distance when the transfer efficiency is 50%:(7)R06=8.8×10-25k2N-4ϕJ,where k2 is the spatial orientation factor of the dipole, N is the refractive index of the medium, Φ is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. J is given by (8)J=∫0∞Fλε(λ)λ4Δλ∫0∞FλΔλ,where F(λ) is the fluorescence intensity of the fluorescent donor of wavelength, λ, and ε(λ) is the molar absorption coefficient of the acceptor at wavelength, λ. In the current study, values of k2 = 2/3, N=1.336, and Φ = 0.15 were used as reported in the literature [50]. Hence, values of J = 1.85 × 10−17 cm3 L mol−1, R0=1.95 nm, E=0.18, and r=2.51 nm were calculated. The donor-to-acceptor distance, r<8 nm [50, 51], indicated that the energy transfer from BSA to EVG occurs with high possibility. Larger BSA-EVG distance, r compared to that of R0 values observed in this study, also reveals the presence of static type quenching mechanism [52, 53]


### 3.5. Conformation Investigation

#### 3.5.1. UV-Vis Absorption Spectra

The complex formation between EVG-BSA was also evident from UV-Vis absorption spectral data (Figure6). It is obvious that the UV absorption intensity of BSA increased regularly with the variation of EVG concentration, indicating that the peptide strands of BSA molecules extended more upon the addition of EVG [51].Figure 6
Absorbance spectra of EVG 10μg mL−1 (A), BSA 0.8 mg mL−1 (B), and EVG-BSA system at EVG concentration of (C) 3.0 μg mL−1, (D) 5.0 μg mL−1, (E) 7.0 μg mL−1, and (F) 10 μg mL−1.


#### 3.5.2. Synchronous Fluorescence

The synchronous fluorescence spectra were measured, shown in Figure7. In the synchronous spectra, the sensitivity associated with fluorescence is maintained while offering several advantages: spectral simplification, spectral bandwidth reduction, and avoidance of different perturbing effects. It shows the tyrosine residues (Tyr) and tryptophan residues (Trp) of BSA when the wavelength interval Δλ is 15 nm and Δλ is 60 nm, respectively [54, 55]. It is obvious that the emission strengths of both tyrosine and tryptophan decreased. A slight blue shift can be observed in Figure 7(b) which indicates that the polarity around tryptophan residue decreased. That is to say, tryptophan residues were placed in a more hydrophobic environment and less exposed to the solvent. Additionally, it can be seen from Figure 7 that a stronger fluorescence quenching effect of tryptophan residues compared with the tyrosine residues after EVG is added. It indicates that the binding site of EVG may be closer to tryptophan than that of the tyrosine residues.Figure 7
Effect of EVG on the synchronous fluorescence spectra of BSA withΔλ = 15 nm (A) and Δλ = 60 nm (B). cBSA = 50 μg·mL−1, cEVG (A–L) = (0, 0.3, 0.7, 1.0, 3.0, 5.0, 10, 12, and 15 μg·mL−1).
(a)
(b)


#### 3.5.3. Three-Dimensional Fluorescence

Moreover, three-dimensional (3D) fluorescence spectra were recorded, and some characteristic 3D parameters are reported in Table4. Two typical fluorescence peaks can easily be observed in the three-dimensional fluorescence spectra Figures 8 and 9. The peak at (224→333) (Peak 1) mainly exhibits the fluorescence characteristic of n→π* transition of the polypeptide backbone of protein structure, C=O [56]. The second peak at (280→337) (Peak 2) revealed the spectral characteristic of Trp and Tyr residues [19]. The Rayleigh scattering peaks and two typical fluorescence peaks can be easily observed in 3D fluorescence contour map of BSA (Figure 9). It is clear that both BSA’s fluorescence peaks were quenched by the addition of EVG, differing in their extents (in the presence of EVG, the intensity ratio of the fluorescence peak to the second order one is 1.776 : 1 whereas it is 1.564 : 1 in the absence of EVG). Additionally, Rayleigh scattering peaks were enhanced that also give clear evidence of complex formation between EVG and BSA [57].Table 4
Three-dimensional fluorescence characteristic parameters of EVG-BSA binding.
BSA
EVG-BSA
Peak 1
Peak 2
Peak 1
Peak 2
Peak position(λex/λem, nm/nm)
224/333
280/337
224/336.5
280/336
Relative intensity (IF)
7352.52
4700.98
4426.11
2491.21
Stokes shift (Δλ/nm)
109
57
112.5
56Figure 8
Three-dimensional spectra of BSA (50μg·mL−1) in the absence (a) and presence (b) of EVG (10 μg·mL−1).
(a)
(b)Figure 9
Contour fluorescence intensity spectra of BSA (a) and EVG-BSA system (b).
(a)
(b)


## 3.1. Fluorescence Quenching of BSA Induced by EVG

Spectroscopic techniques, particularly fluorescence spectroscopy, are able to provide enormous amount of information on the ligand protein binding, such as type, mechanism and strength of the binding, and so forth. It has been previously established that quenching of fluorescence intensity, that is, fluorescence intensity reduction of a certain fluorophore, can occur via several mechanisms including but not limited to formation of a nonfluorescent complex and energy transfer, and so forth [15, 36, 37]. The fluorescence spectra of BSA in presence of different amounts of EVG were measured in the range of 288–500 nm upon excitation at 280 nm. EVG led to a concentration dependent quenching of the intrinsic fluorescence of BSA (Figures 2 and 3) without changing the emission maximum but with a slight change in peak shape at higher EVG concentrations (>10 μg mL−1). These results indicated that an interaction between EVG and BSA is taking place [38].Figure 2
Fluorescence spectra of BSA (50μg·mL−1) in the absence (A) and in presence of EVG at different concentrations. EVG concentrations were 0.1 μg·mL−1 (B), 0.3 μg·mL−1 (C), 0.5 μg·mL−1 (D), 0.7 μg·mL−1 (E), 1.0 μg·mL−1 (F), 3.0 μg·mL−1 (G), 5 μg·mL−1 (H), 7.0 μg·mL−1 (I), 10 μg·mL−1(J), 12 μg·mL−1 (K), and 15 μg·mL−1 (L).Figure 3
The Stern-Volmer (a) and Lineweaver-Burk (b) curves at different temperatures.
(a)
(b)Furthermore, the fluorescence quenching data were analyzed by the Stern-Volmer equation (1) [39] and Lineweaver-Burk equation (2) [40]. Consider(1)F0F=1+KSVQ=1+Kqτ0Q(2)F0-F-1=F0-1+KLB-1F0-1Q-1,where F0 and F are the steady-state fluorescence intensities of BSA in the absence and presence of EVG, respectively, KSV the Stern-Volmer quenching constant and Q is the concentration of quencher (EVG). Kq is the quenching rate constant, τ0 is the average lifetime of the protein without the quencher and KLB is the static quenching constant. Within certain concentration, the curve of F0/F versus Q (Stern-Volmer curve) would be linear if the quenching type is single static or dynamic quenching [17]; similarly, the curve of F0-F-1 versus Q-1 (Lineweaver-Burk curve) would be linear [41] for static quenching. If the quenching type is combined quenching (both static and dynamic), the Stern-Volmer plot is an upward curvature [42]. One way to distinguish dynamic from static quenching is to examine the temperature effect on the interaction of the drug to BSA. The Stern-Volmer and Lineweaver-Burk curves of EVG-BSA at different temperatures are shown in Figure 5. It can be seen that both Stern-Volmer and Lineweaver-Burk curves were linear, which illustrated that the quenching type was probably a static quenching.Moreover, Table1 demonstrates the gradual reduction of KSV and KLB values upon temperature increase that supports the idea of a static type quenching of BSA fluorescence [42]. This hypothesis was also backed up by the Kq values (quenching rate constant), which was computed using the following equation (3)Kq=KSVτ0.Since the fluorescence lifetime of the biopolymer (τ0) can be taken as 10−8 s−1 [43], then the Kq values summarized in Table 1 were calculated for EVG-BSA system to be in the order of 1012 L Mol−1 s−1. Previous reports have shown that the maximum scatter collision quenching constant, Kq of various quenchers with the biopolymer is 2 × 1010 L M−1 s−1 [43]. Ultimately, the Kq values of protein quenching procedure initiated by EVG are higher than the Kq of the scattered procedure. This indicated that the quenching is not initiated by dynamic collision but from the formation of a complex [41].Table 1
Stern-Volmer and Lineweaver-Burk correlation parameters for BSA-EVG interaction.
Temperature (T) (K)
Stern-Volmer parameters*
Lineweaver-Burk parameters*
K
SV  ×  104 (L mol−1)
K
q  ×  1012 (L mol−1 s−1)
r
2
K
LB  ×  105 (L mol−1)
r
2
288
4.13 ± 0.24
4.13
0.9890
4.66 ± 0.15
0.9949
298
4.03 ± 0.27
4.03
0.9867
3.14 ± 0.14
0.9979
309
3.91 ± 0.26
3.91
0.9879
2.43 ± 0.14
0.9983
*Average of four determinations.


## 3.2. Binding Constants and the Number of Binding Sites

Based upon the probability of a static quenching process is taking place between BSA and EVG and if it is assumed that there are independent binding sites to a set of equivalent sites on the protein, the apparent binding constant(K) and the number of binding sites (n) can be determined from [42, 44](4)log⁡F0-FF=log⁡⁡K+nlog⁡⁡Q,where K is the binding constant, reflecting the reaction degree of BSA and EVG; n is the number of binding sites, specifying the number of EVG bound to a BSA molecule. Thus, a plot of Log(F0-F)/F versus log Q could be used to determine K and n. The K and n at different temperatures are summarized in Table 2, indicating good assumptions made for deriving (4). Table 2 also demonstrates a decrease in the binding constant with the increase in temperature, yielding a less stable EVG-BSA complex. Additionally, n values were found to be ~1, which in turn may infer the involvement of one independent class of binding sites on BSA for EVG.Table 2
Binding constantK and the number of binding sites n.
Temperature (T) (K)
K  ×  103 (L mol−1)
n
288
2.68
0.67 ± 0.03
298
1.99
0.72 ± 0.02
309
1.12
0.75 ± 0.03


## 3.3. Thermodynamics and Interaction Forces between EVG and BSA

As the binding constant is dependent on temperature; hence, a thermodynamic process was regarded responsible for the complex formation. Therefore, temperature dependent thermodynamic parameters were determined for further investigating the acting forces between EVG and BSA. Generally, the force between a small molecule and a biological macromolecule includes hydrophobic interaction, hydrogen bonding, van der Waals force, and electrostatic force. The thermodynamic parameters associated with the different types of interactions were previously reported [45]. In which, hydrophobic force may increase ΔHθ and ΔSθ of a system, while van der Waals power may decrease them with negative ΔHθ indicative for hydrogen bonding and electrostatic force usually makes ΔHθ~0 and ΔSθ>0 [46]. Thermodynamic parameters, enthalpy (ΔHθ), and entropy (ΔSθ) of EVG-BSA interaction are significant to confirm binding mode. Binding studies were performed at 288, 298, and 309 K, at which BSA does not undergo any structural degradation. The thermodynamic parameters were evaluated using the Van’t Hoff and Gibbs-Helmholtz [47, 48] equations:(5)log⁡⁡K=-ΔHθ2.303RT+ΔSθ2.303RΔGθ=ΔHθ-T·ΔSθ,where K and R are the binding constant and gas constant, respectively. ΔHθ, ΔGθ, and ΔSθ are enthalpy change, free energy change, and entropy change, respectively. Therefore, a plot of  log⁡K versus 1/T (Figure 4) enables the determination of ΔHθ, ΔGθ, and ΔSθ by (5). Thermodynamic parameters for EVG-BSA binding are summarized in Table 3 and demonstrate that hydrogen bonding and hydrophobic interactions play a role in the binding between EVG and BSA.Table 3
Thermodynamic parameters of BSA-ELV binding.
Temperature (T) (K)
Δ
G
θ (kJ mol−1)
Δ
H
θ (kJ mol−1)
Δ
S
θ (J mol−1 K−1)
288
−42.64
−30.83
41.02
298
−43.05
309
−43.50Figure 4
Van’t Hoff plot for the binding between EVG and BSA, variation of lnK as a function of 1/T.Figure 5
The overlap of the fluorescence spectrum of BSA (a) and the absorbance spectrum of EVG (b).


## 3.4. Energy Transfer between EVG and BSA

The aforementioned spectral studies have suggested that EVG forms a nonfluorescent complex with BSA. Three types of intrinsic fluorophores are known to be found in BSA sequence, namely, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe); however the intrinsic fluorescence of BSA is mainly due to tryptophan [49]. The efficiency of energy transfer can be used to determine the distance, r, between the drug and the tryptophan residues in the protein. The distance r is an average value between the bound EVG and the two tryptophan residues of BSA (Trp-135 and Trp-214) and can be determined using Förster’s resonance energy transfer (FRET) [35, 44]. Generally, energy transfer occurs whenever the emission spectrum of a fluorophore (donor) overlaps with the absorption spectrum of another molecule (acceptor) and the distance between them is less than 8 nm. The overlap of the UV absorption spectrum of EVG with the fluorescence emission spectra of BSA is shown in Figure 5. The efficiency of energy transfer, E, is calculated using (6)E=1-FF0=R06R06+r6,where F and F0 are the fluorescence intensities of BSA in the presence and absence of EVG, r is the distance between acceptor and donor and R0 is the critical distance when the transfer efficiency is 50%:(7)R06=8.8×10-25k2N-4ϕJ,where k2 is the spatial orientation factor of the dipole, N is the refractive index of the medium, Φ is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. J is given by (8)J=∫0∞Fλε(λ)λ4Δλ∫0∞FλΔλ,where F(λ) is the fluorescence intensity of the fluorescent donor of wavelength, λ, and ε(λ) is the molar absorption coefficient of the acceptor at wavelength, λ. In the current study, values of k2 = 2/3, N=1.336, and Φ = 0.15 were used as reported in the literature [50]. Hence, values of J = 1.85 × 10−17 cm3 L mol−1, R0=1.95 nm, E=0.18, and r=2.51 nm were calculated. The donor-to-acceptor distance, r<8 nm [50, 51], indicated that the energy transfer from BSA to EVG occurs with high possibility. Larger BSA-EVG distance, r compared to that of R0 values observed in this study, also reveals the presence of static type quenching mechanism [52, 53]


## 3.5. Conformation Investigation

### 3.5.1. UV-Vis Absorption Spectra

The complex formation between EVG-BSA was also evident from UV-Vis absorption spectral data (Figure6). It is obvious that the UV absorption intensity of BSA increased regularly with the variation of EVG concentration, indicating that the peptide strands of BSA molecules extended more upon the addition of EVG [51].Figure 6
Absorbance spectra of EVG 10μg mL−1 (A), BSA 0.8 mg mL−1 (B), and EVG-BSA system at EVG concentration of (C) 3.0 μg mL−1, (D) 5.0 μg mL−1, (E) 7.0 μg mL−1, and (F) 10 μg mL−1.


### 3.5.2. Synchronous Fluorescence

The synchronous fluorescence spectra were measured, shown in Figure7. In the synchronous spectra, the sensitivity associated with fluorescence is maintained while offering several advantages: spectral simplification, spectral bandwidth reduction, and avoidance of different perturbing effects. It shows the tyrosine residues (Tyr) and tryptophan residues (Trp) of BSA when the wavelength interval Δλ is 15 nm and Δλ is 60 nm, respectively [54, 55]. It is obvious that the emission strengths of both tyrosine and tryptophan decreased. A slight blue shift can be observed in Figure 7(b) which indicates that the polarity around tryptophan residue decreased. That is to say, tryptophan residues were placed in a more hydrophobic environment and less exposed to the solvent. Additionally, it can be seen from Figure 7 that a stronger fluorescence quenching effect of tryptophan residues compared with the tyrosine residues after EVG is added. It indicates that the binding site of EVG may be closer to tryptophan than that of the tyrosine residues.Figure 7
Effect of EVG on the synchronous fluorescence spectra of BSA withΔλ = 15 nm (A) and Δλ = 60 nm (B). cBSA = 50 μg·mL−1, cEVG (A–L) = (0, 0.3, 0.7, 1.0, 3.0, 5.0, 10, 12, and 15 μg·mL−1).
(a)
(b)


### 3.5.3. Three-Dimensional Fluorescence

Moreover, three-dimensional (3D) fluorescence spectra were recorded, and some characteristic 3D parameters are reported in Table4. Two typical fluorescence peaks can easily be observed in the three-dimensional fluorescence spectra Figures 8 and 9. The peak at (224→333) (Peak 1) mainly exhibits the fluorescence characteristic of n→π* transition of the polypeptide backbone of protein structure, C=O [56]. The second peak at (280→337) (Peak 2) revealed the spectral characteristic of Trp and Tyr residues [19]. The Rayleigh scattering peaks and two typical fluorescence peaks can be easily observed in 3D fluorescence contour map of BSA (Figure 9). It is clear that both BSA’s fluorescence peaks were quenched by the addition of EVG, differing in their extents (in the presence of EVG, the intensity ratio of the fluorescence peak to the second order one is 1.776 : 1 whereas it is 1.564 : 1 in the absence of EVG). Additionally, Rayleigh scattering peaks were enhanced that also give clear evidence of complex formation between EVG and BSA [57].Table 4
Three-dimensional fluorescence characteristic parameters of EVG-BSA binding.
BSA
EVG-BSA
Peak 1
Peak 2
Peak 1
Peak 2
Peak position(λex/λem, nm/nm)
224/333
280/337
224/336.5
280/336
Relative intensity (IF)
7352.52
4700.98
4426.11
2491.21
Stokes shift (Δλ/nm)
109
57
112.5
56Figure 8
Three-dimensional spectra of BSA (50μg·mL−1) in the absence (a) and presence (b) of EVG (10 μg·mL−1).
(a)
(b)Figure 9
Contour fluorescence intensity spectra of BSA (a) and EVG-BSA system (b).
(a)
(b)


## 3.5.1. UV-Vis Absorption Spectra

The complex formation between EVG-BSA was also evident from UV-Vis absorption spectral data (Figure6). It is obvious that the UV absorption intensity of BSA increased regularly with the variation of EVG concentration, indicating that the peptide strands of BSA molecules extended more upon the addition of EVG [51].Figure 6
Absorbance spectra of EVG 10μg mL−1 (A), BSA 0.8 mg mL−1 (B), and EVG-BSA system at EVG concentration of (C) 3.0 μg mL−1, (D) 5.0 μg mL−1, (E) 7.0 μg mL−1, and (F) 10 μg mL−1.


## 3.5.2. Synchronous Fluorescence

The synchronous fluorescence spectra were measured, shown in Figure7. In the synchronous spectra, the sensitivity associated with fluorescence is maintained while offering several advantages: spectral simplification, spectral bandwidth reduction, and avoidance of different perturbing effects. It shows the tyrosine residues (Tyr) and tryptophan residues (Trp) of BSA when the wavelength interval Δλ is 15 nm and Δλ is 60 nm, respectively [54, 55]. It is obvious that the emission strengths of both tyrosine and tryptophan decreased. A slight blue shift can be observed in Figure 7(b) which indicates that the polarity around tryptophan residue decreased. That is to say, tryptophan residues were placed in a more hydrophobic environment and less exposed to the solvent. Additionally, it can be seen from Figure 7 that a stronger fluorescence quenching effect of tryptophan residues compared with the tyrosine residues after EVG is added. It indicates that the binding site of EVG may be closer to tryptophan than that of the tyrosine residues.Figure 7
Effect of EVG on the synchronous fluorescence spectra of BSA withΔλ = 15 nm (A) and Δλ = 60 nm (B). cBSA = 50 μg·mL−1, cEVG (A–L) = (0, 0.3, 0.7, 1.0, 3.0, 5.0, 10, 12, and 15 μg·mL−1).
(a)
(b)


## 3.5.3. Three-Dimensional Fluorescence

Moreover, three-dimensional (3D) fluorescence spectra were recorded, and some characteristic 3D parameters are reported in Table4. Two typical fluorescence peaks can easily be observed in the three-dimensional fluorescence spectra Figures 8 and 9. The peak at (224→333) (Peak 1) mainly exhibits the fluorescence characteristic of n→π* transition of the polypeptide backbone of protein structure, C=O [56]. The second peak at (280→337) (Peak 2) revealed the spectral characteristic of Trp and Tyr residues [19]. The Rayleigh scattering peaks and two typical fluorescence peaks can be easily observed in 3D fluorescence contour map of BSA (Figure 9). It is clear that both BSA’s fluorescence peaks were quenched by the addition of EVG, differing in their extents (in the presence of EVG, the intensity ratio of the fluorescence peak to the second order one is 1.776 : 1 whereas it is 1.564 : 1 in the absence of EVG). Additionally, Rayleigh scattering peaks were enhanced that also give clear evidence of complex formation between EVG and BSA [57].Table 4
Three-dimensional fluorescence characteristic parameters of EVG-BSA binding.
BSA
EVG-BSA
Peak 1
Peak 2
Peak 1
Peak 2
Peak position(λex/λem, nm/nm)
224/333
280/337
224/336.5
280/336
Relative intensity (IF)
7352.52
4700.98
4426.11
2491.21
Stokes shift (Δλ/nm)
109
57
112.5
56Figure 8
Three-dimensional spectra of BSA (50μg·mL−1) in the absence (a) and presence (b) of EVG (10 μg·mL−1).
(a)
(b)Figure 9
Contour fluorescence intensity spectra of BSA (a) and EVG-BSA system (b).
(a)
(b)


## 4. Conclusions

In this work we have employed a set of fluorescence spectroscopic UV-visible spectrophotometric analyses to study the interactions of BSA with EVG. EVG proved to bind to BSA resulting in reduction of its fluorescence intensity via static binding. We have computed the binding parameters, and conformational changes leading to increased stability of EVG-BSA complex. Data analysis showed that only one binding site on BSA is available for EVG with a binding constant in the order of 103 L mol−1. Thermodynamic parameters were also computed to be negativefree energy and enthalpy while positive entropy referring a spontaneous reaction of BSA with EVG with both hydrogen bonding and hydrophobic interactions probably involved in the binding. Conformation study showed that the polarity around tryptophan residue decreased. This demonstrates that tryptophan residues were placed in a more hydrophobic microenvironment and less exposed to the solvent. The binding distance between the donor (BSA) and the acceptor (EVG) was calculated based on the Förster’s theory of nonradiation energy transfer.



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*Source: 435674-2015-03-24.xml*