Characterization and antioxidant activity of bovine serum albumin and sulforaphane complex in different solvent systems

Journal of Luminescence 146 (2014) 351–357

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Characterization and antioxidant activity of bovine serum albumin and sulforaphane complex in different solvent systems Xueyan Dong, Rui Zhou, Hao Jing n College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghua East Road, Haidian District, Beijing 100083, China

art ic l e i nf o

a b s t r a c t

Article history: Received 8 May 2013 Received in revised form 12 September 2013 Accepted 27 September 2013 Available online 15 October 2013

Modes and influencing factors of bovine serum albumin (BSA) and sulforaphane (SFN) interaction will help us understand the interaction mechanisms and functional changes of bioactive small molecule and biomacromolecule. This study investigated interaction mechanisms of BSA and SFN and associated antioxidant activity in three solvent systems of deionized water (dH2O), dimethyl sulfoxide (DMSO) and ethanol (EtOH), using Fourier transform infrared spectroscopy (FT-IR), fluorescence spectroscopy, synchronous fluorescence spectroscopy, DPPH and ABTS radical scavenging assays. The results revealed that SFN had ability to quench BSA's fluorescence in static modes, and to interact with BSA at both tyrosine (Tyr) and tryptophan (Trp) residues, while the Trp residues were highly sensitive, which was demonstrated by fluorescence at 340 nm. Hydrophobic forces, hydrogen bonds and van der Waals interactions were all involved in BSA and SFN interaction, which were not significantly changed by three solvents. The binding constant values and binding site numbers were in a descending order of dH2O 4DMSO4 EtOH. The values of free energy change were in a descending order of dH2O 4 DMSO 4EtOH, which indicated that the binding forces were in a descending order of dH2O 4DMSO 4 EtOH. There was no significant difference in antioxidant activity between SFN and BSA–SFN. Moreover, three solvents had not significant influence on antioxidant activity of SFN and BSA–SFN. Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.

Keywords: Bovine serum albumin Sulforaphane Fourier transform infrared spectroscopy Fluorescence spectroscopy Synchronous fluorescence spectroscopy Antioxidant activity

1. Introduction Bovine serum albumin (BSA) plays an important role in the disposition and transportation of various nutrients in blood plasma, and can interact well with many small molecules [1]. BSA has 583 amino acid residues in a single polypeptide chain, among them tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues are considered as intrinsic fluorophores of BSA. While in practical measurement, the intrinsic fluorescence of BSA is mainly contributed by Trp and Tyr residues, because the quantum yield of the Phe residue is very low [2]. The V-shaped BSA molecule consists of three homologous α-helical domains (I, II, and III), and each domain contains two subdomains (A and B), named IA, IB, IIA, IIB, IIIA and IIIB [3]. BSA contains 35 cysteine (Cys) residues, 34 of which are covalently linked to form 17 disulfide bonds whereas only one free thiol group exists at Cys-34 residue [4]. The thiol groups of the Cys residues could be involved in covalent bounding of BSA and electrophilic compounds, such as

Abbreviations: BSA, bovine serum albumin; SFN, sulforaphane; dH2O, deionized water; DMSO, dimethyl sulfoxide; EtOH, ethanol; FT-IR, Fourier transform infrared spectroscopy; Tyr, tyrosine; Trp, tryptophan; DPPH, 1-diphenyl-2-picrylhydrazyl; ABTS, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt n Corresponding author. Tel./fax: 86 10 6273 7909. E-mail addresses: [email protected], [email protected] (H. Jing).

phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), which was associated with their bioactivity [5]. There are 20 Tyr residues and two Trp residues in BSA. Trp-134 is in the IB domain, and is located on the surface of the BSA. Trp-212 is in the IIA domain, and is located within a hydrophobic binding pocket of BSA [6,7]. The Tyr and Trp residues are also important as the binding sites, due to their fluorescence significant [6]. Interaction and mechanism of SFN with Tyr and Trp residues need to be further explored. Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is derived from glucoraphanin. Glucoraphanin, a major member of glucosinolate family, could be hydrolyzed by endogenous myrosinase to form isothiocyanates, mainly SFN [8,9]. There are over 120 glucosinolates in the various varieties of cruciferous vegetables such as broccoli or broccoli sprouts, cauliflower, cabbage and kale [10]. SFN can reverse preneoplastic lesions and has a promising anti-cancer effect on the cancers of lung, colon, prostate, breast, gastric and skin [11,12]. The mechanisms of SFN's bioactivity could be related to activate NF-E2-related factor-2 (Nrf2) and induce the expression of Nrf2-dependent phase-II enzymes such as heme oxygenase-1 (HO-1), NAD(P)H, quinone reductase 1, glutathione reductase and glutathione peroxidase [13]. SFN has two functional groups of SQO and NQCQS, which make it susceptible to degradation under oxygen, heating and alkaline conditions [14,15]. SFN was readily degraded in an aqueous solution at high temperatures of 50 and 100 1C. Its SQO group could

0022-2313/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.09.078

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X. Dong et al. / Journal of Luminescence 146 (2014) 351–357

participate in the formation of epoxide compound, 4-methylthio-4hydroxybutyl isothiocyanate, which went through dehydration and produced 4-isothiocyanato-1-(methylthio)-1-butene. The NQCQS group of SFN could be hydrolyzed and released an amine, which reacted further with SFN to generate N,N′-di-(methylsulfinyl) butyl thiourea [14]. Stability of bioactive small molecule could be improved by interaction with protein. In presence of casein and whey protein, stability of bog bilberry anthocyanin extract (BBAE) had been improved, with less change in the absorbance and antioxidant activity of BBAE [16]. The stability and the antioxidant activity of quercetin, kaempferol and rutin were also preserved by interacting with BSA [17]. The interaction of bioactive small molecule and protein is influenced by many factors, such as pH, ionic strength, temperature, and also solvents [7,18]. Fluorescence quenching and associated binding constants of cochineal red A and BSA complex were significantly reduced by addition of ethanol [19]. The binding constant of isoniazid to BSA was increased with increasing DMSO concentrations from 0% to 10% (v/v), and then decreased when DMSO concentrations were increased from 10% to 20% (v/v). The maximum binding of isoniazid to BSA occurred at the concentration of 10% (v/v) DMSO [20]. While the influence of ethanol and DMSO on the interaction between BSA and SFN and associated contribution to the antioxidant activity of SFN were not clear. The present study investigated the influence of dH2O, DMSO and EtOH as solvent components on the characteristics of BSA and SFN interaction, especially interaction mode of SFN and BSA with Tyr and Trp residues, using FT-IR, fluorescence spectroscopy, synchronous fluorescence spectroscopy, DPPH and ABTS radical scavenging assays.

pressed into a 13-mm diameter disc by applying 15 Torr pressure for 5 min. FT-IR spectra were obtained in the wave number range from 450 to 4000 cm  1 during 100 scans, with 2 cm  1 resolution. 2.4. Fluorescence measurement The fluorescence intensities were recorded with an F-7000 fluorophotometer (Hitachi, Japan). The concentration of BSA was at 1.5
10  5 mol L  1, and SFN concentrations were 0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, 12.0, 13.5 and 15.0
10  4 mol L  1. The fluorescence experiments have been carried out at 25 1C (298 K), 31 1C (304 K) and 37 1C (310 K). An excitation wavelength of 280 nm was selective in all cases for selective excitation of the Trp residues of BSA, and emission spectra are recorded from 280 to 440 nm. The fluorescence quenching data can be analyzed using Stern– Volmer equation [21] F 0 =F ¼ 1 þ K SV ½SFN

ð1Þ

where F0 and F are the fluorescence intensities of BSA without and with the existence of quencher, respectively. KSV is the Stern– Volmer quenching constant with units of L mol  1; [SFN] is in the concentration of mol L  1. Stern–Volmer equation was applied to determine KSV by linear regression of a plot of F0/F against [SFN]. The binding constant (K) and binding sites (n) are calculated by the double-logarithm equation for static quenching [22] lg ½ðF 0  FÞ=F ¼ lg K þ n lg ½SFN

ð2Þ

Binding data at different temperatures were used to analyze the thermodynamic parameters using the van’t Hoff equation [23] ln K ¼  ΔH=ðRTÞ þ ΔS=R

ð3Þ

2. Materials and methods

ΔG ¼  RT ln K

ð4Þ

2.1. Materials

ΔS ¼ ðΔH  ΔGÞ=T

ð5Þ

BSA (fraction V, purity 98%) (A-0332) was purchased from AMRESCO (Amresco Inc., OH, USA). SFN (purity 95%, HPLC grade) was purchased from College of Life Science and Technology, Beijing University of Chemical Technology (Beijing, China). DPPH (D9132), ABTS (A-1888) and DMSO (purity 99.5%) were all purchased from Sigma-Aldrich, Inc. (St. Louis, MO). EtOH (purity 99.9%) was purchased from Xilong Chemical Co., Ltd. (Beijing, China). All other reagents were of analytical grade. 2.2. Preparation of the complex of BSA–SFN The stock solution of BSA (1.5
10  3 mol L  1) was prepared with deionized water (dH2O, 18 MΩ). The stock solution of SFN (1.5
10  2 mol L  1) was prepared with dH2O. Both stock solutions were stored in the refrigerator at 4 1C prior to use. The BSA stock solution was diluted before use with dH2O (pH 6.82), 10% DMSO (v/v, pH 6.28) and 10% EtOH (v/v, pH 6.26). The BSA–SFN complex was prepared by adding SFN stock solution into diluted BSA stock solution. The final concentration of BSA was 1.5
10  5 mol L  1, and the concentrations of SFN were 0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, 12.0, 13.5 and 15.0
10  4 mol L  1. 2.3. FT-IR measurement FT-IR spectra of pure BSA and BSA–SFN complexes were recorded using a Spectrum 100 FT-IR spectrometer (Perkin-Elmer, USA), equipped with a deuterated triglycine sulfate (DTGS) detector. The spectrometer was continuously purged with dried air. 2 mg of freezedried sample was mixed with 200 mg KBr and ground gently with an agate pestle and mortar under an infrared lamp and afterwards was

where K is the association constant, T is the absolute temperature, R is the gas constant (8.3145 J mol  1 K  1), ΔH is the enthalpy change, ΔS is the entropy change and ΔG is free energy change. 2.5. Synchronous fluorescence measurement Synchronous fluorescence spectra of the samples were obtained after scanning them in the wavelength range of 260– 320 nm and 240–320 nm for the difference between excitation and emission wavelengths (Δλ) of 15 and 60 nm, respectively. The final concentration of BSA in each sample was 1.5
10  5 mol L  1 while SFN concentrations were 0, 1.5, 3.0, 6.0, 9.0, 12.0, 15.0
10  4 mol L  1. 2.6. Antioxidant activity assessment The concentrations of SFN and BSA were set at 1.5
10  3 mol L  1 and 1.5
10  6 mol L  1, respectively. 2.6.1. DPPH assay The DPPH free radical scavenging activity was determined according to the method of Fang et al. [24]. Stock solutions of DPPH were prepared at 2.5 mmol L  1 and then diluted with ethanol to 0.15 mmol L  1. Each sample (15 μL) was mixed with 0.05 mol L  1 (pH 7.4) Tris–HCl buffer (60 μL) and 0.15 mmol L  1 DPPH working solution (150 μL) in a 96-well plate. The mixture was shaken vigorously and then left to stand for 30 min in the dark. The absorbance (Asample) at 517 nm was recorded using a microplate reader (model 680, Bio-Rad Laboratories, Inc., Hercules, CA).

X. Dong et al. / Journal of Luminescence 146 (2014) 351–357

2.6.2. ABTS assay The experiment was carried out by the method proposed by Fang et al. [24]. Briefly, 140 mmol L  1 ABTS stock solution was diluted with water to 14 mmol L  1. Five hundred microlitres of 14 mmol L  1 ABTS dilution and 500 μL of 4.9 mmol L  1 potassium persulfate (KPS) solution were mixed throughly, and then it was left to stand in the dark and at room temperature for at least 12 h to obtain ABTS. The ABTS was then diluted with PBS to an absorbance of 0.70 70.02 at 734 nm before use. After addition of 90 μL of diluted ABTS solution to 10 μL of samples, the absorbance (Asample) reading was taken exactly after 4 min. All of the samples were analyzed in triplicate. The absorbance of the color (Acolor) was obtained by replacing the DPPH or ABTS with ethanol or PBS, respectively. The absorbance of the blank (Ablank) was obtained by replacing the sample with dH2O, 10% DMSO or 10% EtOH. The radical scavenging rate (% RSR) for both DPPH and ABTS was calculated using following equation: RSRð%Þ ¼ ½Ablank  ðAsample  Acolor Þ=Ablank
100%

ð6Þ

2.7. Statistical analysis All experiments were performed in triplicate. The data were presented as the mean 7SD. Means were compared by one-way ANOVA, followed by Tukey's pairwise comparisons, using Minitab software (Minitab Inc., State College, PA). The level of confidence required for significance was set at po 0.05.

3. Results and discussion 3.1. FT-IR spectra of BSA–SFN complex

amide II, respectively; in 10% EtOH from 1657 to 1644 cm  1 and 1539 to 1558 cm  1 for amide I and amide II, respectively. The negative features for amide I and positive features for amide II bands were similar in dH2O, 10% DMSO and 10% EtOH. The IR vibration bands corresponding to the amide Ι (80% CQO stretch, near 1600–1700 cm  1) and amide II (60% N–H bend and 40% C–N stretch, near 1500–1600 cm  1) are generally employed to study secondary structures of protein [25]. The changes for amide I and amide II bands were due to SFN interact with the CQO, C–N and N–N groups in the BSA polypeptides and resulted in the rearrangement of the polypeptide carbonyl hydrogenbonding network [26]. The results indicated that a complex formation between BSA and SFN and hydrogen bonds involved in the interaction between BSA and SFN, and all of them were consistent in dH2O, 10% DMSO and 10% EtOH. 3.2. BSA–SFN binding parameters In the presence of SFN, the fluorescence intensity of BSA was significant decreased, but the peak at 340 nm had no obvious shift. The results indicated that SFN could interact with BSA and quench its intrinsic fluorescence. The pattern of changes on the fluorescence spectra of BSA was consistent in dH2O, 10% DMSO and 10% EtOH (Fig. 2). Theoretically, BSA has three potential intrinsic fluorophores, including Tyr, Trp and Phe [2]. The quantum yield of the Phe residue is too low to be detected. Tyr and Trp residues have emission fluorescence at 304 nm and 340 nm, respectively. Only the fluorescence at the emission wavelength of 340 nm could be detected for Trp residues, due to possible overlap of emission fluorescence of Tyr and Trp residues. Trp residues are mainly responsible for intrinsic fluorescence of BSA. The fluorescence intensity at 340 nm was significant decreased with the increasing of SFN concentration, which indicated that interaction occurred at the Trp residues. Fluorescence of the Trp residues changes of BSA were investigated at different concentrations (10–70%, v/v) of DMSO and EtOH aqueous solutions. The results showed that the fluorescent intensity of BSA decreased with increasing concentrations of DMSO and

%T

%T

In Fig. 1, curve a is the spectrum of free BSA, and curve b shows the spectrum of BSA after the addition of SFN, which were obtained in dH2O, 10% DMSO or 10% EtOH. The peak shifts were observed in dH2O from 1657 to 1656 cm  1 and from 1539 to 1543 cm  1 for amide I and amide II, respectively; in 10% DMSO from 1657 to 1649 cm  1 and 1539 to 1545 cm  1 for amide I and

353

3950 3450 2950 2450 1950 1450

950

3950 3450 2950 2450 1950 1450

450

cm-1

950

450

%T

cm-1

3950 3450 2950 2450 1950 1450

950

450

cm-1 Fig. 1. FT-IR spectra of BSA in the absence (curve a) and presence (curve b) of SFN in different solvent systems. (A) dH2O, (B) 10% DMSO, and (C) 10% EtOH. CBSA: 1.5
10  5 mol L  1 and CSFN: 9.0
10  4 mol L  1.

X. Dong et al. / Journal of Luminescence 146 (2014) 351–357

500

A: dH2O SFN/BSA (molar ratio)

400 300 200 100 0 280

320

360

400

Fluorescence Intensity (a.u.)

Fluorescence Intensity (a.u.)

500

SFN/BSA (molar ratio)

400 300 200 100 0 280

440

500

B: 10%DMSO

320

Wavelength (nm)

360

400

Fluorescence Intensity (a.u.)

354

C: 10%EtOH

SFN/BSA (molar ratio)

400 300 200 100 0 280

440

320

Wavelength (nm)

360

400

440

Wavelength (nm)

Fig. 2. Fluorescence emission spectra of BSA in presence of SFN with different concentrations in different solvent systems. (A) dH2O, (B) 10% DMSO, and (C) 10% EtOH. The molar ratio of SFN to BSA (SFN/BSA) was 0–10. CBSA: 1.5
10  5 mol L  1; CSFN: 0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, 12.0, 13.5 and 15.0
10  4 mol L  1. (T ¼298 K).

1.3

1.3

A: dH2O

-298 K -304 K -310 K

1.3

-298 K -304 K -310 K

C: 10%EtOH

-298 K -304 K -310 K

1.2

1.1

1

F0/F

1.2

F0/F

F0/F

1.2

B: 10%DMSO

1.1

1.1

1

1

0.9

0.9

0.9 0

3

6

9

12

15

0

[SFN] (10-4mol L-1)

3

6

9

12

0

15

3

6

9

12

15

[SFN] (10-4mol L-1)

[SFN] (10-4mol L-1)

Fig. 3. Modified Stern–Volmer plots of BSA and SFN at different temperatures in different solvent systems. (A) dH2O, (B) 10% DMSO, and (C) 10% EtOH. F0 and F represent the fluorescence intensities of BSA in the absence and in the presence of SFN. [SFN] represents the concentration of sulforaphane. CBSA: 1.5
10  5 mol L  1; CSFN: 0, 1.5, 3.0, 6.0, 9.0, 12.0, and 15.0
10  4 mol L  1. (T ¼298, 304, and 310 K).

Table 1 Modified Stern–Volmer equations and quenching constants at different temperatures in different solvent systems. Solvent sys.

T (K)

Stern–Volmer equations, F0/F¼ 1þ KSV [SFN]

KSV (L mol  1)

2

2

Correlation coefficient (r)

dH2O

298 304 310

F0/F ¼1.45
10 [SFN] þ1.0096 F0/F ¼1.32
102 [SFN] þ1.0033 F0/F ¼1.21
102 [SFN] þ 0.9947

1.45
10 1.32
102 1.21
102

0.9951 0.9993 0.9990

10% DMSO

298 304 310

F0/F ¼1.17
102 [SFN]þ 1.0114 F0/F ¼0.99
102 [SFN] þ1.0076 F0/F ¼0.89
102 [SFN] þ1.0002

1.17
102 0.99
102 0.89
102

0.9972 0.9944 0.9969

10% EtOH

298 304 310

F0/F ¼1.10
102 [SFN] þ 1.0111 F0/F ¼0.94
102 [SFN] þ1.0085 F0/F ¼0.85
102 [SFN] þ0.9993

1.10
102 0.94
102 0.85
102

0.9995 0.9980 0.9977

EtOH. The influence of DMSO and EtOH on the fluorescence intensity and peak position of BSA was less at the concentration of 10% (v/v) (data not shown). It had been known that BSA could be denatured by DMSO and EtOH at high concentrations, whereas BSA remained in the native state at the low concentrations (up to 10%, v/v) [27,28]. Therefore, 10% DMSO and 10% EtOH were chosen as the solvent systems in our experiments. Fig. 3 shows the Stern–Volmer plots for the BSA–SFN systems, and the corresponding values of quenching constants (KSV) at different temperatures were also listed in Table 1. An inverse correlation between KSV values and temperatures was noticed, and the KSV values were in a descending order of dH2O 410% DMSO 410% EtOH. It is in static mode if the quenching rate constant (KSV) decreases with increasing temperature. On the

contrary, the reverse effect is observed with dynamic quenching [29]. It suggested that fluorescence quenching of BSA by SFN was in static mode, which was the same in three solvent systems. Fig. 4 displays the double logarithmic linear dependence of lg(F0  F)/F versus lg[SFN], and the corresponding values of binding constants (K) and binding sites (n) at different temperatures were also listed in Table 2. The n values were approximately equal to 1, and K values were decreased with the increasing temperature. Both K and n values were found in a descending order of dH2O410% DMSO410% EtOH. The polarity of three solvent solutions was in a descending order of dH2O (10.2)4DMSO (7.2)4EtOH (4.3). Yan et al. [30] reported DMSO and EtOH have lower polarity than dH2O, which would increase the solubility of the SFN, therefore reduce the binding of BSA and SFN as shown in Table 2.

X. Dong et al. / Journal of Luminescence 146 (2014) 351–357

-2

-2 A: dH2O -1.8

■ -298 K -304 K ▲ -310 K

-1.8

-1.4 -1.2

-1.6

-1.4 -1.2

-1.4 -1.2

-1

-1

-1

-0.8

-0.8

-0.8

-0.6 -2.8

-3.2

-3.6

-4

-0.6 -2.8

lg[SFN]

■-298 K -304 K ▲-310 K

C: 10%EtOH

-1.8

lg (F0-F) / F

lg (F0-F) / F

lg (F0-F) / F

-2

B: 10%DMSO ■ -298 K -304 K ▲ -310 K

-1.6

-1.6

355

-3.2

-3.6

-4

-0.6 -2.8

-3.2

-4

-3.6

lg[SFN]

lg[SFN]

Fig. 4. The plots of lg [(F0  F)/F] versus lg[SFN] for SFN binding to BSA at different temperatures in different solvent systems. (A) dH2O, (B) 10% DMSO, and (C) 10% EtOH. F0 and F represent the fluorescence intensities of BSA in the absence and in the presence of SFN. [SFN] represents the concentration of sulforaphane. CBSA: 1.5
10  5 mol L  1; CSFN: 0, 1.5, 3.0, 6.0, 9.0, 12.0, and 15.0
10  4 mol L  1.

Table 2 The binding constants and binding sites of BSA–SFN at different temperatures in different solvent systems. Solvent sys.

T (K)

lg(F0  F)/F¼ lg Kþ n lg [SFN]

KSV (L mol  1)

n

Correlation coefficient (r)

dH2O

298 304 310

lg(F0  F)/F¼ 2.2105þ 1.0463 lg [SFN] lg(F0  F)/F¼ 1.8935þ 0.9224 lg [SFN] lg(F0  F)/F¼ 1.6204 þ0.8176 lg [SFN]

162.368 78.253 41.725

1.0463 0.9224 0.8176

0.9972 0.9979 0.9958

10% DMSO

298 304 310

lg(F0  F)/F¼ 1.9736 þ1.0018 lg [SFN] lg(F0  F)/F¼ 1.6204 þ0.8621 lg [SFN] lg(F0  F)/F¼ 1.1864 þ0.6988 lg [SFN]

94.102 41.725 15.360

1.0018 0.8620 0.6988

0.9993 0.9980 0.9977

10% EtOH

298 304 310

lg(F0  F)/F¼ 1.8729þ 0.9534 lg [SFN] lg(F0  F)/F¼ 1.4859 þ0.8148 lg [SFN] lg(F0  F)/F¼ 0.9212þ 0.6133 lg [SFN]

74.628 23.613 8.341

0.9534 0.8148 0.6133

0.9964 0.9911 0.9930

3.3. Thermodynamic parameters The ΔH, ΔS and ΔG were all in negative values in three solvent systems (Table 3). Negative values of ΔS and ΔH were associated with hydrogen bonds and van der Waals interactions, and the positive values were related with hydrophobic forces [31]. Therefore, interaction of BSA and SFN was through the hydrogen bonds and van der Waals interactions, which was not significantly changed in three solvent systems. FT-IR spectroscopy also demonstrated that hydrogen bonds involved in the interaction between BSA and SFN. The negative value for ΔG indicated that the interaction process was spontaneous. The higher ΔG absolute value indicated stronger binding [19]. Absolute ΔG values (for example at 298 K) and the binding forces were found in a descending order of dH2O (12.611) 410% DMSO (11.260) 410% EtOH (10.685). 3.4. Synchronous fluorescence spectra The fluorescence intensity of Tyr residues at 288 nm was decreased with increasing SFN concentration. No obvious wavelength shift of the emission maxima (288 nm) was observed upon addition of SFN. The pattern of changes of synchronous fluorescence spectra (Δλ was 15 nm) of BSA was consistent in dH2O, 10% DMSO and 10% EtOH (Fig. 5-A1, B1, and C1). The fluorescence intensity of Trp residues at 278 nm was decreased with increasing SFN concentration, and the pattern of changes was similar in dH2O, 10% DMSO and 10% EtOH. Whereas, synchronous fluorescence spectra obtained with Δλ¼60 nm showed a red shift from 276 to 278 nm after SFN addition, which was observed in

Table 3 Thermodynamics constants of BSA–SFN at different temperatures in different solvent systems. Solvent sys.

T (K)

ΔH (kJ mol  1)

ΔS (J mol  1)

ΔG (kJ mol  1)

dH2O

298 304 310

 86.907

 249.316  249.629  249.325

 12.611  11.020  9.617

298 304 310

 116.206

 352.169  351.234  352.144

 11.260  9.431  7.041

298 304 310

 140.644

 436.103  434.197  436.054

 10.685  8.648  5.467

10% DMSO

10% EtOH

10% DMSO and 10% EtOH, but not in dH2O (Fig. 5-A2, B2, and C2). The fluorescence spectra of Tyr and Trp residues could be detected by synchronous fluorescence, with the peak values at 288 nm for Tyr residues and 278 nm for Trp residues. The fluorescence intensity at 288 nm and 278 nm was significant decreased with the increasing of SFN concentration, which indicated that both Tyr and Trp residues involved in the interaction of BSA and SFN. The hydrophobicity of Tyr residues had no remarkable change during the binding process in three solvent systems. While the hydrophobicity of Trp residues decreased in 10% DMSO and 10% EtOH, but no obvious change in dH2O. The results indicated that SFN interacted with both Tyr and Trp residues of BSA through hydrophobic forces, and the strength of hydrophobic forces was in a descending order of dH2O4 10% DMSO 410% EtOH.

356

X. Dong et al. / Journal of Luminescence 146 (2014) 351–357

Fig. 5. Synchronous fluorescence spectra of BSA in presence of SFN with different concentrations in different solvent systems. (A1, A2) dH2O; (B1, B2) 10% DMSO; and (C1, C2) 10% EtOH. SFN/BSA: the molar concentration ratio of SFN to BSA. CBSA: 1.5
10  5 mol L  1; CSFN: 0, 1.5, 3.0, 6.0, 9.0, 12.0, and 15.0
10  4 mol L  1.

3.5. Antioxidant activity of SFN and BSA–SFN SFN was used at the concentration of 1.5
10  3 mol L  1 in the DPPH and ABTS radical scavenging assays. The DPPH radical scavenging rates of SFN and BSA–SFN were 6% and 5.6%, respectively (Fig. 6-A). The ABTS radical scavenging rates of SFN and BSA–SFN were 21% and 20%, respectively (Fig. 6-B). There was no significant difference between SFN and BSA–SFN in both DPPH and ABTS radical scavenging rates, and no significant difference was observed in three solvent systems for DPPH or ABTS radical scavenging rates. SFN exhibited the weak antioxidant activity with 50% inhibitory concentration (IC50) of 1.0
10  2 mol L  1 in DPPH scavenging assay [32]. The low DPPH radical scavenging rates were also reported for glucoerucin and erucin [32]. Our results indicated that the active groups of SFN for antioxidant activity were not

interfered by BSA. BSA and SFN interaction happened mainly between the SFN groups (SQO and NQCQS) and BSA groups (CQO, C–N, and N–H) [33].

4. Conclusions SFN had ability to quench BSA's fluorescence, and to interact with BSA at both Tyr and Trp residues. Hydrophobic forces, hydrogen bonds and van der Waals interactions were all involved in BSA and SFN interaction. The binding constant values, binding site numbers and binding forces between BSA and SFN were influenced by different solvents, with a descending order of dH2O4DMSO4 EtOH. It could be attributed to polarity of the solvents, which was also in a descending order of dH2O4DMSO4EtOH. While solvent

DPPH RSR (%)

X. Dong et al. / Journal of Luminescence 146 (2014) 351–357

357

a

Fig. 6. DPPH and ABTS scavenging activity of SFN and BSA–SFN in different solvent systems. CBSA: 1.5
10  5 mol L  1 and CSFN: 1.5
10  3 mol L  1. RSRBSA–SFN ¼RSR(BSA–SFN)  RSRBSA; RSRBSA–SFN: the DPPH and ABTS scavenging activities of SFN in BSA–SFN; RSR(BSA–SFN): the DPPH and ABTS scavenging activities of BSA–SFN complex; and RSRBSA: the DPPH and ABTS scavenging activities of BSA. Different letters in the figure denote that the mean difference is significant at po0.05.

type had no significant effect on antioxidant activity of SFN and BSA–SFN. The present study quantified binding affinity of SFN to BSA, and demonstrated solvents effect on the binding mode of SFN and BSA. It provides insight into the interaction mechanism of bioactive small molecule and macromolecule of SFN and BSA. Further study will be carried out on the physical and chemical property of BSA– SFN nanoparticle and its stability under physiological condition. Acknowledgments This research was supported by the National Science and Technology Support Program (No. 31171676) and Chinese Universities Scientific Fund (2012YJ083). References [1] Y.L. Xiang, F.Y. Wu, Spectrochim. Acta, Part A 77 (2010) 430. [2] M.R. Ganjali, F. Faridbod, A. Divsalar, A.A. Saboury, P. Norouzi, G. Rezaei Behbehani, S. Abdolahzadeh, Int. J. Electrochem. Sci. 5 (2010) 852. [3] B.X. Huang, H.Y. Kim, J. Am. Soc. Mass Spectrom. 15 (2004) 1237. [4] I. Ascone, L. Messorl, A. Caslnl, C. Gabblanl, A. Balerna, F. Dell’Unto, A.C. Castellano, Inorg. Chem. 47 (2008) 8629. [5] L.X. Mi, X.T. Wang, S. Govind, B.L. Hood, T.D. Veenstra, T.P. Conrads, D.T. Saha, R. Goldman, F.L. Chung, Cancer Res. 67 (2007) 6409. [6] X.L. Han, F.F. Tian, Y.S. Ge, F.L. Jiang, L. Lai, D.W. Li, Q.L.Y. Yu, J. Wang, C. Lin, Y. Liu, J. Photochem. Photobiol. B 109 (2012) 1. [7] T.H. Wang, Z.M. Zhao, B.Z. Wei, L. Zhang, L. Ji, J. Mol. Struct. 970 (2010) 128. [8] P.A. Egner, T.W. Kensler, J.G. Chen, S.J. Gange, J.D. Groopman, M.D. Friesen, Chem. Res. Toxicol. 21 (2008) 1991.

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