International Journal of Biological Macromolecules 78 (2015) 333–338
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Interaction of bovine serum albumin with starch nanoparticles prepared by TEMPO-mediated oxidation Haoran Fan a,b , Na Ji a , Mei Zhao a , Liu Xiong a , Qingjie Sun a,∗ a b
School of Food Science and Engineering, Qingdao Agricultural University, Qingdao, PR China The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, PR China
a r t i c l e
i n f o
Article history: Received 6 March 2015 Received in revised form 4 April 2015 Accepted 14 April 2015 Available online 20 April 2015 Keywords: Starch nanoparticles Protein Interaction Spectroscopy Circular dichroism
a b s t r a c t The objective of this study was to elucidate the interaction of starch nanoparticles prepared through TEMPO oxidation (TEMPO-SNPs) with protein (bovine serum albumin) by various spectroscopic techniques and transmission electron microscopy (TEM). The enhanced absorbance observed by UV spectra and the decrease in fluorescence spectroscopy of bovine serum albumin (BSA) induced by TEMPO-SNPs demonstrated the occurrence of an interaction between BSA and TEMPO-SNPs. The quenching constant was inversely correlated with temperature, showing that the quenching effect of TEMPO-SNPs was static quenching. Electrostatic force had a leading contribution to the binding roles of BSA on TEMPO-SNPs, which was confirmed by negative enthalpy change and positive entropy change. When interacting with TEMPO-SNPs at different concentrations, the content of the ␣-helix structure in BSA decreased and sheet and random coil structures increased, indicating that TEMPO-SNPs had an effect on the secondary conformation of BSA. Furthermore, TEM images suggested that nanoparticle-protein complexes were formed. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanoparticles have attracted great interest in recent years due to their unique physical and chemical properties, and potential for application in food, packaging, and containers [1–3]. Starch has been considered one of the most promising biodegradable materials because it is a versatile biopolymer with immense potential and can be obtained at a low price for use in industries [4,5]. Starch nanoparticles (SNPs) are nano-sized (1–1000 nm) particulates of starch prepared by mechanical treatment such as ultrasonication [6], extrusion [7], high-pressure homogenization [8], enzymatic treatment [9,10] and acid hydrolysis [11–13]. When a nanomaterial enters a physiological environment, its surface is immediately covered by a layer of proteins, forming what is known as the protein ‘corona’ [14,15]. As water soluble proteins, serum albumin is the most abundant and basic protein of blood plasma. It is mainly involved in the transport of substances in the blood that are poorly soluble in water, including drugs, and also in the disposition of endogenous and exogenous compounds present in blood [16,17]. Albumins are also reported to associate with nanoscale particles to promote their transportation through the cells [18,19]. In this study, BSA has been taken as a model protein because of its major physiological significance, structural homology ∗ Corresponding author. Tel.: +86 532 88030448; fax: +86 532 88030449. E-mail address: [email protected] (Q. Sun). http://dx.doi.org/10.1016/j.ijbiomac.2015.04.028 0141-8130/© 2015 Elsevier B.V. All rights reserved.
to human serum albumin (HSA), ready availability, and its soluble nature in water [20]. Since nanoparticle usage in the biomedical field is increasing, it is necessary to understand their interaction with biomolecules such as BSA. Many researchers have reported the interaction of BSA with metal NPs such as Ag [21], CdS [22], Au [23], ZnO [24] and polymer NPs such as chitosan [25], polymeric [26], hyperbranched poly (amine) ester [27], polystyrene [28], and polymer [29]. SNPs were widely used in applications such as tire mixtures [30], nanocomposite films [31,32], and biomedicine as a nanocarrier [33]; it is essential to study their interactions with the proteins in a biological system. To the best of our knowledge, there is no prior report demonstrating the interaction of BSA with SNPs. We previously prepared 20–60 nm TEMPO-SNPs by ultrasonic-assisted oxidation [6]. Therefore, the aim of our present study was to investigate the structural changes in BSA upon interaction with TEMPO-SNPs through various spectroscopic techniques such as UV–Visible, fluorescence, and circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM). 2. Materials and methods 2.1. Materials Waxy maize starch (WMS; approximately 1% amylose and 99% amylopectin) was obtained from Zhucheng Xingmao Corn
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Development Co., Ltd. (Shangdong, China). Sodium hypochlorite (Sinopharm Chemical Regent Co. Ltd., China) containing 10 g active chlorine/100 g was used in this experiment. Bovine serum albumin pH 7.0 (BSA: MW 66 kDa and >95% purity) was procured from Sigma–Aldrich (St. Louis, MO). All other chemicals used were of analytical grade. 2.2. Preparation of TEMPO-SNPs As described by Sun et al. [6], TEMPO-SNPs were prepared by ultrasonic treatment with TEMPO oxidation. A slurry of 10% WMS (100 g) was put in a beaker and held at 5 ◦ C. The TEMPO (0.048 g; 0.01 mol per anhydroglucose unit of starch) and sodium bromide (0.635 g; 0.2 mol per anhydroglucose unit of starch) were dissolved in 100 ml distilled water. After TEMPO was completely dissolved, the solution was added to the starch solution at 5 ◦ C. The pH of the solution was adjusted to 9.5 with 0.5 M NaOH. Then, 20 g of sodium hypochlorite solution was slowly added to the starch solution, and the pH was maintained at 9.5 by continuous addition of 0.5 M NaOH. The beaker was then dipped into the ultrasonic bath (KQ-500TDE, Kunshan, China) with power ultrasound of 500 W with 100% amplitude at a frequency of 40 kHz. Sonication was continued for 180 min under continuous stirring at 5 ◦ C. The mixture was separated by centrifugation at 50 s−1 for 10 min, and the supernatant was centrifuged at 166.67 s−1 for 10 min. The precipitate was then washed with water and centrifuged three times; then the precipitate was lyophilized to obtain TEMPO-SNPs. 2.3. Interaction of BSA with TEMPO-SNPs Various concentrations of TEMPO-SNPs (0.2, 0.4, 0.6, 0.8, and 1.0 × 10−9 mol/L) were interacted with a constant BSA (1.5 × 10−5 mol/L) concentration for 30 min on a rotary shaker (300 rpm) at room temperature [34]. After the interaction period, characterization studies were performed with the help of UV–Visible spectroscopy, fluorescence spectroscopy, CD spectroscopy, and TEM. Molar concentration of TEMPO-SNPs is hypothesized from the molecular numbers of TEMPO-SNPs multiplied by Avogadro’s constant. The molecular numbers of TEMPO-SNPs were calculated by dividing the sample weight by the weight of one TEMPO-SNP. The weight of one TEMPO-SNP was calculated by the following equation: m = v
wavelength of 290 nm and emission spectra in the range of 300–500 nm. 2.6. Circular dichroism (CD) spectroscopy Circular dichroism (CD) spectroscopy was performed to analyze the changes that occurred in the secondary structure of BSA by TEMPO-SNPs. For CD analysis, BSA was interacted with various concentrations of TEMPO-SNPs (0.2, 0.4, 0.6, 0.8, and 1.0 × 10−9 mol/L) in Milli-Q water. After 30 min of incubation, CD spectra were analyzed using CD spectroscopy (JASCO J-715). Changes to the secondary structure of the protein were confirmed by comparing the CD spectrum data of the control sample to the test sample. 2.7. Transmission electron microscopy (TEM) Images of interaction between BSA and TEMPO-SNPs were examined using a transmission electron micrograph (TEM, HT-7700, Hitachi Instruments Ltd., Japan). A drop of BSA (1.5 × 10−5 mol/L) conjugated TEMPO-SNPs (0.6 × 10−9 mol/L) was placed on a copper grid and lyophilized. 3. Results and discussion 3.1. UV–Vis absorption spectra The UV–Vis absorption spectra of BSA in absence and presence of TEMPO-SNPs are shown in Fig. 1. The UV–Vis spectroscopy for BSA showed an adsorption maximum (max ) at 278 nm which was attributed to the –* electron transition of the BSA molecule [35]. The UV absorption spectra intensity of BSA at 278 nm gradually strengthened along with the increasing concentration of TEMPOSNPs with no shift. Therefore, the results demonstrated the increase in hydrophobicity in the micro-environment surrounded by amino acid residues. Gradual exposure of indole nitrogen in Trp (tryptophan), and phenolate oxygen in Tyr (tyrosine) residues in a hydrophobic situation with TEMPO-SNPs led to the changes in UV spectra of BSA. Kathiravan et al. [36] reported a similar enhanced absorbance of BSA upon the addition of ZnO NPs. They suggested that enhanced absorbance was due to the interaction of colloidal ZnO NPs with BSA through static quenching mechanisms. Xiao et al. [27] reported that the addition of HPAE led to a gradual decrease in the absorption intensity of BSA.
(1)
Where is the density of starch, v is the volume of TEMPO-SNPs, and the average particle size of TEMPO-SNPs is 50 nm.
0.8
-5
BSA(1.5×10 mol/L) -9 BSA+TEMPO-SNPs(0.2×10 mol/L) -9 BSA+TEMPO-SNPs(0.4×10 mol/L) -9 BSA+TEMPO-SNPs(0.6×10 mol/L) -9 BSA+TEMPO-SNPs(0.8×10 mol/L) -9 BSA+TEMPO-SNPs(1.0×10 mol/L)
0.7
2.4. UV-Visible spectroscopy
0.5
Absorbance
After the interaction, 3 mL of sample was used to record UVVisible spectra using a UV-Visible spectrophotometer (Shimadzu2600, Japan) from 250 to 350 nm. The spectra obtained from the system were analyzed to evaluate the changes occurring in the absorption peak of BSA at 278 nm, a characteristic peak of BSA [20].
0.6
0.4 0.3 0.2
2.5. Fluorescence spectra analysis
0.1
Fluorescence of BSA after interaction with TEMPO-SNPs was measured using a spectrofluorometer (SL174, ELICO). BSA (1.5 × 10−5 mol/L) was interacted with various concentrations of TEMPO-SNPs (0.2, 0.4, 0.6, 0.8, and 1.0 × 10−9 mol/L) and 3 mL of the sample was subjected to analysis at the excitation
0.0 260
280
300
320
340
Wavelength (nm) Fig. 1. The UV–Vis spectra of BSA-conjugated TEMPO-SNPs from 1 to 6: CBSA = 1.5 × 10−5 mol/L; CTEMPO-SNPs /(10−9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
H. Fan et al. / International Journal of Biological Macromolecules 78 (2015) 333–338 -5
BSA(1.5×10 mol/L) -9 BSA+TEMPO-SNPs(0.2×10 mol/L) -9 BSA+TEMPO-SNPs(0.4×10 mol/L) -9 BSA+TEMPO-SNPs(0.6×10 mol/L) -9 BSA+TEMPO-SNPs(0.8×10 mol/L) -9 BSA+TEMPO-SNPs(1.0×10 mol/L)
2500
Intensity
2000 1500
335
Table 1 The thermodynamic parameters of the TEMPO-SNPs and BSA binding procedure. T/K
Ka/ n Ksv / (108 L mol−1 ) (108 L mol−1 )
H/ kJ mol−1
G/ kJ mol−1
S/ J mol−1 K−1
293 303 310
1.824 1.482 1.472
−40.05 −40.05 −40.05
−48.13 −47.17 −46.37
27.59 23.59 20.38
3.82 1.85 0.75
1.5168 1.6181 1.7594
1000 500 0 300
350
400
450
500
Wavelength (nm) Fig. 2. The fluorescence quenching spectra of BSA-conjugated TEMPO-SNPs from 1 to 6: CBSA = 1.5 × 10−5 mol/L; CTEMPO-SNPs /(10−9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0. Fig. 4. Double-log plot of TEMPO-SNPs’ effect on BSA fluorescence.
3.2. Fluorescence measurement of BSA The fluorescence emission spectrum of BSA was measured in the presence of TEMPO-SNPs, and the mechanism of fluorescence quenching was analyzed. The fluorescence spectra of BSA in the presence of TEMPO-SNPs at different concentrations are shown in Fig. 2. As can be seen from Fig. 2, the maximum fluorescence emission wavelength of BSA was 345 nm, and the fluorescence intensity of BSA showed a significant gradual decrease along with the increase of TEMPO-SNPs. The intrinsic fluorescence of BSA is mainly contributed by its two tryptophan (Trp) moieties-Trp 212 (deeply buried in the hydrophobic loop) and Trp 134 (more exposed to hydrophilic environment), which is very sensitive to the local environment [37]. It indicated that quenching of the intrinsic BSA fluorescence by TEMPO-SNPs occurred. Therefore, we concluded from the fluorescence results that the TEMPO-SNPs and BSA complexed. Several previous works on the interaction of BSA with Ag nanoparticles [38] has also reported similar quenching. 3.3. Mechanism of fluorescence quenching
The ratio of F0 /F, plotted against TEMO-SNPs concentration according to Equation (1) at different temperatures, is presented in Fig. 3. Fluorescence quenching could appear as a result of interactions including molecular collisions (dynamic quenching), complex formation (static quenching), energy transfer, and conformational changes [39]. As can be seen from Fig. 3, F0 /F has a linear correlation to the concentration of TEMPO-SNPs and KSV values that are listed in Table 1. The KSV values of different temperatures were 1.824 × 108 L/mol (293 K), 1.482 × 108 L/mol (303 K), and 1.472 × 108 L/mol (310 K), respectively. The results showed that KSV values varied according to temperature, with KSV values decreasing as the temperature increased. As we know, dynamic quenching depends on diffusion, and the coefficient of diffusion will augment with the increase of temperature [41]. However, for static quenching, the stability of complex formations will decrease with the increase of temperature, so the quenching constant is inversely correlated with temperature [27]. Therefore, these results confirmed that the quenching mechanism was a static quenching rather than a dynamic quenching mechanism. Similar
Fluorescence quenching is described by the Stern-Volmer equation [39]: F0 /F = 1 + KSV [Q] = 1 + Kq 0 [Q]
(2)
1.2
293K
1.15
303K
1.1
310K
60 40 20
CD (mdeg)
Where F0 and F are the fluorescence intensity without and with quencher (TEMPO-SNPs), respectively. KSV is the Stern-Volmer quenching constant, [Q] is the concentration of quencher, Kq is the quenching rate constant, and 0 is the average lifetime of molecules in the absence of quencher and its value is about 10−8 s [40].
F0 /F
-5
BSA(1.5×10 mol/L) -9 BSA+TEMPO-SNPs(0.2×10 mol/L) -9 BSA+TEMPO-SNPs(0.4×10 mol/L) -9 BSA+TEMPO-SNPs(0.6×10 mol/L) -9 BSA+TEMPO-SNPs(0.8×10 mol/L) -9 BSA+TEMPO-SNPs(1.0×10 mol/L)
0 -20 -40
1.05
-60
1 0.95
180 0.0
0.2
0.4
0.6
0.8
1.0
-9
190
200
210
220
230
240
250
260
270
Wavelength (nm)
[Q](10 mol /L) Fig. 3. The Stern–Volmer curves of BSA quenched by TEMPO-SNPs at 293, 303, and 310 K.
Fig. 5. The circular dichroism spectra of the TEMPO-SNPs and BSA binding procedure from 1 to 6: CBSA = 1.5 × 10−5 mol/L; CTEMPO-SNPs /(10−9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
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Table 2 The changes of BSA secondary-structure in the TEMPO-SNPs and BSA binding process. Sample
␣-helix (%)
-sheet (%)
Random coil (%)
BSA BSA + 0.2TEMPO-SNPs BSA + 0.4TEMPO-SNPs BSA + 0.6TEMPO-SNPs BSA + 0.8TEMPO-SNPs BSA + 1.0TEMPO-SNPs
56 49 43 36 33 15
15 18 20 24 25 33
29 33 37 40 42 52
observations were also reported by Li et al. [42] on the interaction of BSA with amidated single-walled carbon nanotubes, who described the significance of fluorescence quenching as an indicator of static quenching mechanisms. Kathiravan et al. [36] reported that enhanced absorbance was due to the interaction of colloidal ZnO NPs with BSA through static quenching mechanisms. Xiao et al. [27] also reported that the mechanism of fluorescence quenching of polymer-hyperbranched poly (amine) ester with BSA was static quenching. 3.4. Binding constant and binding sites For the static quenching interaction, it is assumed that there are independent binding sites in the BSA, the binding constant (Ka) and the number sites (n) can be estimated from the double logarithm regression curve of log(F0 − F)/F versus lg[Q] based on the following equation: lg
F − F 0 F
= lgKa + nlg[Q]
(3)
Where F0 and F are the fluorescence intensity without and with quencher (TEMPO-SNPs), respectively. Ka is the binding constant, n is the number of binding sites, and [Q] is the concentration of quencher. Fig. 4 shows the double logarithm regression curves of lg (F0 − F)/F versus lg [Q] at 293, 303, and 310 K. Observing the changes in lg (F0 − F)/F with lg [Q], values of K and n are listed in Table 1. The Ka decrease with rising temperatures suggested that the capacity of TEMPO-SNPs to bind to BSA weakened at higher temperatures. The value of n for the interaction of TEMPO-SNPs and BSA at different temperatures was more than 1, indicating that the binding of BSA to TEMPO-SNPs had exceeded a single binding site, and conclusion supported by findings in a previous reports [43].
3.5. Binding force The binding force between TEMPO-SNPs and BSA could be supported by hydrogen bond force, hydrophobic force, Van der Waals force, and electrostatic force. The thermodynamic parameters are usually applied to judge the categories of binding forces, and can be calculated by the Van’t Hoff equation: G = −RT ln Ka
(4)
H =
RT1 T2 ln(Ka2 /Ka1 ) T2 − T1
(5)
S =
H − G T
(6)
Fig. 6. TEM images (a) pure TEMPO-SNPs (bar = 500 nm), (b) BSA and TEMPO-SNPs complex (bar = 500 nm) and (c) BSA and TEMPO-SNPs complex (bar = 200 nm).
H. Fan et al. / International Journal of Biological Macromolecules 78 (2015) 333–338
Where H, G, and S denote enthalpy change, free enthalpy change, and entropy change, respectively; Ka is the binding constant corresponding to various temperatures; R is the gas constant, and T is experimental temperature. When H < 0 or H = 0 and S > 0, the main binding force is electrostatic force; when H < 0, and S < 0, the main binding force is van der Waals force or hydrogen bond force, and when H > 0, and S > 0, the main binding force is hydrophobic force [44]. Values of H, G, and S are shown in Table 1, and because H < 0 and S > 0, it suggests that the main binding force was electrostatic force. The process of binding was a spontaneous process in which the Gibbs free energy change was negative.
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driven mainly by electrostatic interaction. The process of binding was a spontaneous process in which the Gibbs free energy change was negative. TEM images showed that TEMPO-SNPs and BSA nanoparticles had protein corona, indicating that the TEMPOSNPs and BSA interacted. The ␣-helix decreased from 56% to 15%, and -sheet and random coil increased from 15% and 29%, to 33% and 52%, respectively. This study is expected to provide new insight starch nanoparticles interacting with BSA, and to provide theoretical support for its application in the field of biomedical materials and nano drug carriers.
Acknowledgement
3.6. Circular dichroism spectroscopy The secondary structural changes of BSA affected by the concentration of TEMPO-SNPs are shown in Fig. 5, and quantitative changes in ␣-helix structural content are presented in Table 2. As shown in Fig. 5, there was a distinct decrease in the intensities of the CD signals at 208 and 222 nm, which was a typical characteristic of ␣-helix structure in the CD spectra of BSA with TEMPO-SNPs [45]. The ␣-helix structure in BSA was 56%, an approximate match to previous research [21]. Compared to native BSA, the ␣-helical content decreased when BSA interacted with TEMPO-SNPs at different concentrations, and the decrease in ␣-helix was mainly balanced by an enhancement of -sheet and random coil, indicating that the conformation of BSA became more compact. In addition, the contents of -turn and random coil in BSA were significantly affected by the addition of TEMPO-SNPs. These results indicated that TEMPOSNPs had an affinity with BSA and led to structural change. Similar behavior was reported by Saurabh et al. [34], who found that the CD spectra of BSA conjugated with silver nanoparticles showed considerable changes in the secondary structure of BSA, with an increase in -sheet structure and random structure at the cost of ␣-helix. Rajeshwari et al. [46] found that Al2 O3 NPs decreased the ␣-helix content of BSA from 47.0% to 23.6% when compared with that of the native BSA. Xiao et al. [27] also found that the ␣-helical content of BSA decreased in presence of polymer-hyperbranched poly (amine) ester. 3.7. TEM The size and morphology of TEMPO-SNPs that interacted with BSA were characterized by TEM (Fig. 6). The TEM images showed that the TEMPO-SNPs and TEMPO-SNPs/BSA were nearly spherical or elliptical in shape. Fig. 6a shows the size of TEMPO-SNPs was around 50 nm. More importantly, the TEM micrograph provided clear and direct visual evidence of two different regions in the synthesized bioconjugated nanoparticle. From Fig. 6b and c (enlarge of Fig. 6b), we noticed that the surface of TEMPO-SNPs occupied by BSA suggesting that the BSA was adsorbed in a protein corona on the surface of TEMPO-SNPs. This could be due to the much larger surface area of TEMPO-SNPs being amenable to adsorbing BSA. Hu et al. [47] also reported that BSA formed protein coronas around Fe3 O4 NPs. 4. Conclusions The interaction of TEMPO-SNPs with BSA was investigated using UV-Vis, fluorescence, and CD spectroscopy, and TEM measurements. We observed an increase in the absorbance peak at 278 nm in UV–Vis spectra along with an increasing concentration of TEMPO-SNPs. Fluorescence quenching of BSA with TEMPO-SNPs was static in nature, and the number of binding sites (n) was more than 1. The negative enthalpy change and the positive entropy change indicated that the interaction of TEMPO-SNPs and BSA was
Financial support from the Qingdao Municipal Science and Technology Plan Project (14-2-3-48-nsh) is gratefully acknowledged.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Interaction of bovine serum albumin with starch nanoparticles prepared by TEMPO-mediated oxidation Haoran Fan a,b , Na Ji a , Mei Zhao a , Liu Xiong a , Qingjie Sun a,∗ a b
School of Food Science and Engineering, Qingdao Agricultural University, Qingdao, PR China The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, PR China
a r t i c l e
i n f o
Article history: Received 6 March 2015 Received in revised form 4 April 2015 Accepted 14 April 2015 Available online 20 April 2015 Keywords: Starch nanoparticles Protein Interaction Spectroscopy Circular dichroism
a b s t r a c t The objective of this study was to elucidate the interaction of starch nanoparticles prepared through TEMPO oxidation (TEMPO-SNPs) with protein (bovine serum albumin) by various spectroscopic techniques and transmission electron microscopy (TEM). The enhanced absorbance observed by UV spectra and the decrease in fluorescence spectroscopy of bovine serum albumin (BSA) induced by TEMPO-SNPs demonstrated the occurrence of an interaction between BSA and TEMPO-SNPs. The quenching constant was inversely correlated with temperature, showing that the quenching effect of TEMPO-SNPs was static quenching. Electrostatic force had a leading contribution to the binding roles of BSA on TEMPO-SNPs, which was confirmed by negative enthalpy change and positive entropy change. When interacting with TEMPO-SNPs at different concentrations, the content of the ␣-helix structure in BSA decreased and sheet and random coil structures increased, indicating that TEMPO-SNPs had an effect on the secondary conformation of BSA. Furthermore, TEM images suggested that nanoparticle-protein complexes were formed. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanoparticles have attracted great interest in recent years due to their unique physical and chemical properties, and potential for application in food, packaging, and containers [1–3]. Starch has been considered one of the most promising biodegradable materials because it is a versatile biopolymer with immense potential and can be obtained at a low price for use in industries [4,5]. Starch nanoparticles (SNPs) are nano-sized (1–1000 nm) particulates of starch prepared by mechanical treatment such as ultrasonication [6], extrusion [7], high-pressure homogenization [8], enzymatic treatment [9,10] and acid hydrolysis [11–13]. When a nanomaterial enters a physiological environment, its surface is immediately covered by a layer of proteins, forming what is known as the protein ‘corona’ [14,15]. As water soluble proteins, serum albumin is the most abundant and basic protein of blood plasma. It is mainly involved in the transport of substances in the blood that are poorly soluble in water, including drugs, and also in the disposition of endogenous and exogenous compounds present in blood [16,17]. Albumins are also reported to associate with nanoscale particles to promote their transportation through the cells [18,19]. In this study, BSA has been taken as a model protein because of its major physiological significance, structural homology ∗ Corresponding author. Tel.: +86 532 88030448; fax: +86 532 88030449. E-mail address: [email protected] (Q. Sun). http://dx.doi.org/10.1016/j.ijbiomac.2015.04.028 0141-8130/© 2015 Elsevier B.V. All rights reserved.
to human serum albumin (HSA), ready availability, and its soluble nature in water [20]. Since nanoparticle usage in the biomedical field is increasing, it is necessary to understand their interaction with biomolecules such as BSA. Many researchers have reported the interaction of BSA with metal NPs such as Ag [21], CdS [22], Au [23], ZnO [24] and polymer NPs such as chitosan [25], polymeric [26], hyperbranched poly (amine) ester [27], polystyrene [28], and polymer [29]. SNPs were widely used in applications such as tire mixtures [30], nanocomposite films [31,32], and biomedicine as a nanocarrier [33]; it is essential to study their interactions with the proteins in a biological system. To the best of our knowledge, there is no prior report demonstrating the interaction of BSA with SNPs. We previously prepared 20–60 nm TEMPO-SNPs by ultrasonic-assisted oxidation [6]. Therefore, the aim of our present study was to investigate the structural changes in BSA upon interaction with TEMPO-SNPs through various spectroscopic techniques such as UV–Visible, fluorescence, and circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM). 2. Materials and methods 2.1. Materials Waxy maize starch (WMS; approximately 1% amylose and 99% amylopectin) was obtained from Zhucheng Xingmao Corn
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Development Co., Ltd. (Shangdong, China). Sodium hypochlorite (Sinopharm Chemical Regent Co. Ltd., China) containing 10 g active chlorine/100 g was used in this experiment. Bovine serum albumin pH 7.0 (BSA: MW 66 kDa and >95% purity) was procured from Sigma–Aldrich (St. Louis, MO). All other chemicals used were of analytical grade. 2.2. Preparation of TEMPO-SNPs As described by Sun et al. [6], TEMPO-SNPs were prepared by ultrasonic treatment with TEMPO oxidation. A slurry of 10% WMS (100 g) was put in a beaker and held at 5 ◦ C. The TEMPO (0.048 g; 0.01 mol per anhydroglucose unit of starch) and sodium bromide (0.635 g; 0.2 mol per anhydroglucose unit of starch) were dissolved in 100 ml distilled water. After TEMPO was completely dissolved, the solution was added to the starch solution at 5 ◦ C. The pH of the solution was adjusted to 9.5 with 0.5 M NaOH. Then, 20 g of sodium hypochlorite solution was slowly added to the starch solution, and the pH was maintained at 9.5 by continuous addition of 0.5 M NaOH. The beaker was then dipped into the ultrasonic bath (KQ-500TDE, Kunshan, China) with power ultrasound of 500 W with 100% amplitude at a frequency of 40 kHz. Sonication was continued for 180 min under continuous stirring at 5 ◦ C. The mixture was separated by centrifugation at 50 s−1 for 10 min, and the supernatant was centrifuged at 166.67 s−1 for 10 min. The precipitate was then washed with water and centrifuged three times; then the precipitate was lyophilized to obtain TEMPO-SNPs. 2.3. Interaction of BSA with TEMPO-SNPs Various concentrations of TEMPO-SNPs (0.2, 0.4, 0.6, 0.8, and 1.0 × 10−9 mol/L) were interacted with a constant BSA (1.5 × 10−5 mol/L) concentration for 30 min on a rotary shaker (300 rpm) at room temperature [34]. After the interaction period, characterization studies were performed with the help of UV–Visible spectroscopy, fluorescence spectroscopy, CD spectroscopy, and TEM. Molar concentration of TEMPO-SNPs is hypothesized from the molecular numbers of TEMPO-SNPs multiplied by Avogadro’s constant. The molecular numbers of TEMPO-SNPs were calculated by dividing the sample weight by the weight of one TEMPO-SNP. The weight of one TEMPO-SNP was calculated by the following equation: m = v
wavelength of 290 nm and emission spectra in the range of 300–500 nm. 2.6. Circular dichroism (CD) spectroscopy Circular dichroism (CD) spectroscopy was performed to analyze the changes that occurred in the secondary structure of BSA by TEMPO-SNPs. For CD analysis, BSA was interacted with various concentrations of TEMPO-SNPs (0.2, 0.4, 0.6, 0.8, and 1.0 × 10−9 mol/L) in Milli-Q water. After 30 min of incubation, CD spectra were analyzed using CD spectroscopy (JASCO J-715). Changes to the secondary structure of the protein were confirmed by comparing the CD spectrum data of the control sample to the test sample. 2.7. Transmission electron microscopy (TEM) Images of interaction between BSA and TEMPO-SNPs were examined using a transmission electron micrograph (TEM, HT-7700, Hitachi Instruments Ltd., Japan). A drop of BSA (1.5 × 10−5 mol/L) conjugated TEMPO-SNPs (0.6 × 10−9 mol/L) was placed on a copper grid and lyophilized. 3. Results and discussion 3.1. UV–Vis absorption spectra The UV–Vis absorption spectra of BSA in absence and presence of TEMPO-SNPs are shown in Fig. 1. The UV–Vis spectroscopy for BSA showed an adsorption maximum (max ) at 278 nm which was attributed to the –* electron transition of the BSA molecule [35]. The UV absorption spectra intensity of BSA at 278 nm gradually strengthened along with the increasing concentration of TEMPOSNPs with no shift. Therefore, the results demonstrated the increase in hydrophobicity in the micro-environment surrounded by amino acid residues. Gradual exposure of indole nitrogen in Trp (tryptophan), and phenolate oxygen in Tyr (tyrosine) residues in a hydrophobic situation with TEMPO-SNPs led to the changes in UV spectra of BSA. Kathiravan et al. [36] reported a similar enhanced absorbance of BSA upon the addition of ZnO NPs. They suggested that enhanced absorbance was due to the interaction of colloidal ZnO NPs with BSA through static quenching mechanisms. Xiao et al. [27] reported that the addition of HPAE led to a gradual decrease in the absorption intensity of BSA.
(1)
Where is the density of starch, v is the volume of TEMPO-SNPs, and the average particle size of TEMPO-SNPs is 50 nm.
0.8
-5
BSA(1.5×10 mol/L) -9 BSA+TEMPO-SNPs(0.2×10 mol/L) -9 BSA+TEMPO-SNPs(0.4×10 mol/L) -9 BSA+TEMPO-SNPs(0.6×10 mol/L) -9 BSA+TEMPO-SNPs(0.8×10 mol/L) -9 BSA+TEMPO-SNPs(1.0×10 mol/L)
0.7
2.4. UV-Visible spectroscopy
0.5
Absorbance
After the interaction, 3 mL of sample was used to record UVVisible spectra using a UV-Visible spectrophotometer (Shimadzu2600, Japan) from 250 to 350 nm. The spectra obtained from the system were analyzed to evaluate the changes occurring in the absorption peak of BSA at 278 nm, a characteristic peak of BSA [20].
0.6
0.4 0.3 0.2
2.5. Fluorescence spectra analysis
0.1
Fluorescence of BSA after interaction with TEMPO-SNPs was measured using a spectrofluorometer (SL174, ELICO). BSA (1.5 × 10−5 mol/L) was interacted with various concentrations of TEMPO-SNPs (0.2, 0.4, 0.6, 0.8, and 1.0 × 10−9 mol/L) and 3 mL of the sample was subjected to analysis at the excitation
0.0 260
280
300
320
340
Wavelength (nm) Fig. 1. The UV–Vis spectra of BSA-conjugated TEMPO-SNPs from 1 to 6: CBSA = 1.5 × 10−5 mol/L; CTEMPO-SNPs /(10−9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
H. Fan et al. / International Journal of Biological Macromolecules 78 (2015) 333–338 -5
BSA(1.5×10 mol/L) -9 BSA+TEMPO-SNPs(0.2×10 mol/L) -9 BSA+TEMPO-SNPs(0.4×10 mol/L) -9 BSA+TEMPO-SNPs(0.6×10 mol/L) -9 BSA+TEMPO-SNPs(0.8×10 mol/L) -9 BSA+TEMPO-SNPs(1.0×10 mol/L)
2500
Intensity
2000 1500
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Table 1 The thermodynamic parameters of the TEMPO-SNPs and BSA binding procedure. T/K
Ka/ n Ksv / (108 L mol−1 ) (108 L mol−1 )
H/ kJ mol−1
G/ kJ mol−1
S/ J mol−1 K−1
293 303 310
1.824 1.482 1.472
−40.05 −40.05 −40.05
−48.13 −47.17 −46.37
27.59 23.59 20.38
3.82 1.85 0.75
1.5168 1.6181 1.7594
1000 500 0 300
350
400
450
500
Wavelength (nm) Fig. 2. The fluorescence quenching spectra of BSA-conjugated TEMPO-SNPs from 1 to 6: CBSA = 1.5 × 10−5 mol/L; CTEMPO-SNPs /(10−9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0. Fig. 4. Double-log plot of TEMPO-SNPs’ effect on BSA fluorescence.
3.2. Fluorescence measurement of BSA The fluorescence emission spectrum of BSA was measured in the presence of TEMPO-SNPs, and the mechanism of fluorescence quenching was analyzed. The fluorescence spectra of BSA in the presence of TEMPO-SNPs at different concentrations are shown in Fig. 2. As can be seen from Fig. 2, the maximum fluorescence emission wavelength of BSA was 345 nm, and the fluorescence intensity of BSA showed a significant gradual decrease along with the increase of TEMPO-SNPs. The intrinsic fluorescence of BSA is mainly contributed by its two tryptophan (Trp) moieties-Trp 212 (deeply buried in the hydrophobic loop) and Trp 134 (more exposed to hydrophilic environment), which is very sensitive to the local environment [37]. It indicated that quenching of the intrinsic BSA fluorescence by TEMPO-SNPs occurred. Therefore, we concluded from the fluorescence results that the TEMPO-SNPs and BSA complexed. Several previous works on the interaction of BSA with Ag nanoparticles [38] has also reported similar quenching. 3.3. Mechanism of fluorescence quenching
The ratio of F0 /F, plotted against TEMO-SNPs concentration according to Equation (1) at different temperatures, is presented in Fig. 3. Fluorescence quenching could appear as a result of interactions including molecular collisions (dynamic quenching), complex formation (static quenching), energy transfer, and conformational changes [39]. As can be seen from Fig. 3, F0 /F has a linear correlation to the concentration of TEMPO-SNPs and KSV values that are listed in Table 1. The KSV values of different temperatures were 1.824 × 108 L/mol (293 K), 1.482 × 108 L/mol (303 K), and 1.472 × 108 L/mol (310 K), respectively. The results showed that KSV values varied according to temperature, with KSV values decreasing as the temperature increased. As we know, dynamic quenching depends on diffusion, and the coefficient of diffusion will augment with the increase of temperature [41]. However, for static quenching, the stability of complex formations will decrease with the increase of temperature, so the quenching constant is inversely correlated with temperature [27]. Therefore, these results confirmed that the quenching mechanism was a static quenching rather than a dynamic quenching mechanism. Similar
Fluorescence quenching is described by the Stern-Volmer equation [39]: F0 /F = 1 + KSV [Q] = 1 + Kq 0 [Q]
(2)
1.2
293K
1.15
303K
1.1
310K
60 40 20
CD (mdeg)
Where F0 and F are the fluorescence intensity without and with quencher (TEMPO-SNPs), respectively. KSV is the Stern-Volmer quenching constant, [Q] is the concentration of quencher, Kq is the quenching rate constant, and 0 is the average lifetime of molecules in the absence of quencher and its value is about 10−8 s [40].
F0 /F
-5
BSA(1.5×10 mol/L) -9 BSA+TEMPO-SNPs(0.2×10 mol/L) -9 BSA+TEMPO-SNPs(0.4×10 mol/L) -9 BSA+TEMPO-SNPs(0.6×10 mol/L) -9 BSA+TEMPO-SNPs(0.8×10 mol/L) -9 BSA+TEMPO-SNPs(1.0×10 mol/L)
0 -20 -40
1.05
-60
1 0.95
180 0.0
0.2
0.4
0.6
0.8
1.0
-9
190
200
210
220
230
240
250
260
270
Wavelength (nm)
[Q](10 mol /L) Fig. 3. The Stern–Volmer curves of BSA quenched by TEMPO-SNPs at 293, 303, and 310 K.
Fig. 5. The circular dichroism spectra of the TEMPO-SNPs and BSA binding procedure from 1 to 6: CBSA = 1.5 × 10−5 mol/L; CTEMPO-SNPs /(10−9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
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Table 2 The changes of BSA secondary-structure in the TEMPO-SNPs and BSA binding process. Sample
␣-helix (%)
-sheet (%)
Random coil (%)
BSA BSA + 0.2TEMPO-SNPs BSA + 0.4TEMPO-SNPs BSA + 0.6TEMPO-SNPs BSA + 0.8TEMPO-SNPs BSA + 1.0TEMPO-SNPs
56 49 43 36 33 15
15 18 20 24 25 33
29 33 37 40 42 52
observations were also reported by Li et al. [42] on the interaction of BSA with amidated single-walled carbon nanotubes, who described the significance of fluorescence quenching as an indicator of static quenching mechanisms. Kathiravan et al. [36] reported that enhanced absorbance was due to the interaction of colloidal ZnO NPs with BSA through static quenching mechanisms. Xiao et al. [27] also reported that the mechanism of fluorescence quenching of polymer-hyperbranched poly (amine) ester with BSA was static quenching. 3.4. Binding constant and binding sites For the static quenching interaction, it is assumed that there are independent binding sites in the BSA, the binding constant (Ka) and the number sites (n) can be estimated from the double logarithm regression curve of log(F0 − F)/F versus lg[Q] based on the following equation: lg
F − F 0 F
= lgKa + nlg[Q]
(3)
Where F0 and F are the fluorescence intensity without and with quencher (TEMPO-SNPs), respectively. Ka is the binding constant, n is the number of binding sites, and [Q] is the concentration of quencher. Fig. 4 shows the double logarithm regression curves of lg (F0 − F)/F versus lg [Q] at 293, 303, and 310 K. Observing the changes in lg (F0 − F)/F with lg [Q], values of K and n are listed in Table 1. The Ka decrease with rising temperatures suggested that the capacity of TEMPO-SNPs to bind to BSA weakened at higher temperatures. The value of n for the interaction of TEMPO-SNPs and BSA at different temperatures was more than 1, indicating that the binding of BSA to TEMPO-SNPs had exceeded a single binding site, and conclusion supported by findings in a previous reports [43].
3.5. Binding force The binding force between TEMPO-SNPs and BSA could be supported by hydrogen bond force, hydrophobic force, Van der Waals force, and electrostatic force. The thermodynamic parameters are usually applied to judge the categories of binding forces, and can be calculated by the Van’t Hoff equation: G = −RT ln Ka
(4)
H =
RT1 T2 ln(Ka2 /Ka1 ) T2 − T1
(5)
S =
H − G T
(6)
Fig. 6. TEM images (a) pure TEMPO-SNPs (bar = 500 nm), (b) BSA and TEMPO-SNPs complex (bar = 500 nm) and (c) BSA and TEMPO-SNPs complex (bar = 200 nm).
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Where H, G, and S denote enthalpy change, free enthalpy change, and entropy change, respectively; Ka is the binding constant corresponding to various temperatures; R is the gas constant, and T is experimental temperature. When H < 0 or H = 0 and S > 0, the main binding force is electrostatic force; when H < 0, and S < 0, the main binding force is van der Waals force or hydrogen bond force, and when H > 0, and S > 0, the main binding force is hydrophobic force [44]. Values of H, G, and S are shown in Table 1, and because H < 0 and S > 0, it suggests that the main binding force was electrostatic force. The process of binding was a spontaneous process in which the Gibbs free energy change was negative.
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driven mainly by electrostatic interaction. The process of binding was a spontaneous process in which the Gibbs free energy change was negative. TEM images showed that TEMPO-SNPs and BSA nanoparticles had protein corona, indicating that the TEMPOSNPs and BSA interacted. The ␣-helix decreased from 56% to 15%, and -sheet and random coil increased from 15% and 29%, to 33% and 52%, respectively. This study is expected to provide new insight starch nanoparticles interacting with BSA, and to provide theoretical support for its application in the field of biomedical materials and nano drug carriers.
Acknowledgement
3.6. Circular dichroism spectroscopy The secondary structural changes of BSA affected by the concentration of TEMPO-SNPs are shown in Fig. 5, and quantitative changes in ␣-helix structural content are presented in Table 2. As shown in Fig. 5, there was a distinct decrease in the intensities of the CD signals at 208 and 222 nm, which was a typical characteristic of ␣-helix structure in the CD spectra of BSA with TEMPO-SNPs [45]. The ␣-helix structure in BSA was 56%, an approximate match to previous research [21]. Compared to native BSA, the ␣-helical content decreased when BSA interacted with TEMPO-SNPs at different concentrations, and the decrease in ␣-helix was mainly balanced by an enhancement of -sheet and random coil, indicating that the conformation of BSA became more compact. In addition, the contents of -turn and random coil in BSA were significantly affected by the addition of TEMPO-SNPs. These results indicated that TEMPOSNPs had an affinity with BSA and led to structural change. Similar behavior was reported by Saurabh et al. [34], who found that the CD spectra of BSA conjugated with silver nanoparticles showed considerable changes in the secondary structure of BSA, with an increase in -sheet structure and random structure at the cost of ␣-helix. Rajeshwari et al. [46] found that Al2 O3 NPs decreased the ␣-helix content of BSA from 47.0% to 23.6% when compared with that of the native BSA. Xiao et al. [27] also found that the ␣-helical content of BSA decreased in presence of polymer-hyperbranched poly (amine) ester. 3.7. TEM The size and morphology of TEMPO-SNPs that interacted with BSA were characterized by TEM (Fig. 6). The TEM images showed that the TEMPO-SNPs and TEMPO-SNPs/BSA were nearly spherical or elliptical in shape. Fig. 6a shows the size of TEMPO-SNPs was around 50 nm. More importantly, the TEM micrograph provided clear and direct visual evidence of two different regions in the synthesized bioconjugated nanoparticle. From Fig. 6b and c (enlarge of Fig. 6b), we noticed that the surface of TEMPO-SNPs occupied by BSA suggesting that the BSA was adsorbed in a protein corona on the surface of TEMPO-SNPs. This could be due to the much larger surface area of TEMPO-SNPs being amenable to adsorbing BSA. Hu et al. [47] also reported that BSA formed protein coronas around Fe3 O4 NPs. 4. Conclusions The interaction of TEMPO-SNPs with BSA was investigated using UV-Vis, fluorescence, and CD spectroscopy, and TEM measurements. We observed an increase in the absorbance peak at 278 nm in UV–Vis spectra along with an increasing concentration of TEMPO-SNPs. Fluorescence quenching of BSA with TEMPO-SNPs was static in nature, and the number of binding sites (n) was more than 1. The negative enthalpy change and the positive entropy change indicated that the interaction of TEMPO-SNPs and BSA was
Financial support from the Qingdao Municipal Science and Technology Plan Project (14-2-3-48-nsh) is gratefully acknowledged.
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