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Octyl gallate: An antioxidant demonstrating selective and sensitive fluorescent property
Food Chemistry 219 (2017) 268–273
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Octyl gallate: An antioxidant demonstrating selective and sensitive fluorescent property Qing Wang, Yongkui Zhang, Hui Li ⇑ College of Chemical Engineering, Sichuan University, Chengdu 610065, People’s Republic of China
a r t i c l e
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Article history: Received 7 June 2016 Received in revised form 16 August 2016 Accepted 26 September 2016 Available online 28 September 2016 Chemical compounds studied in this article: PubChem CID: 61253 Keywords: Octyl gallate Fluorescence enhancement Förster resonance energy transfer (FRET) Complex formation Displacement
a b s t r a c t Octyl gallate (OG) is an internationally recognized antioxidant that demonstrates selective and sensitive fluorescent property. The fluorescence of OG can be selectively enhanced in the presence of human serum albumin (HSA) and bovine serum albumin (BSA). The specific structures of HSA and BSA provided the basic conditions for fluorescence enhancement. OG yielded approximately 49- and 11-fold increments in emission intensity in the presence of HSA and BSA at a molar ratio of 1:1, respectively. The lifetimes of HSA and BSA correspondingly decreased. A Förster resonance energy transfer phenomenon occurred during interaction between OG and HSA or BSA. Our in-depth investigation of OG–HSA interaction showed that formation of a stable complex was an important prerequisite to efficiently enhance the fluorescence of OG. The selective and sensitive fluorescent property of OG can possibly be used to determine OG concentration via the standard addition method, which must be performed under certain conditions. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Autoxidation naturally occurs between molecular oxygen and unsaturated lipids in the environment (Shahidi, Janitha, & Wanasundara, 1992). Deterioration of foodstuffs is often prevented by adding lipid soluble antioxidants during food processing (Aruoma, Murcia, Butler, & Halliwell, 1993). Octyl gallate (OG, Fig. 1) is an internationally recognized antioxidant that can be added into foods to inhibit lipid autoxidation (Joint FAO/WHO Expert Committee on Food Additives, 1962). OG has long been evaluated by the Scientific Committee on Food and the Joint FAO/WHO Expert Committee on Food Additives; in addition, OG has been approved in many countries and regions, such as in European Union (EU) and Australia, as a food additive (EFSA ANS Panel, 2015). Fluorescence-based assays involving small molecules that can induce a strong optical signal while interacting with proteins have considerably attracted the interest of researchers (Dey, Gaur, Giri, & Ghosh, 2016; Liu et al., 2016). These probes themselves usually emit no or weak fluorescence, although the fluorescence of these probes can be significantly enhanced when they interact with proteins. Many research groups have developed highly efficient ⇑ Corresponding author. E-mail address: [email protected] (H. Li). http://dx.doi.org/10.1016/j.foodchem.2016.09.157 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
molecular probes to detect proteins, such as human serum albumin (HSA) (Anees, Sreejith, & Ajayaghosh, 2014; He, Zang, James, Li, & Chen, 2015; Zhu, Du, Cao, Fan, & Peng, 2016). Interestingly, fluorescence enhancement does not occur in a specific site in HSA. Crystallographic analyses have revealed that HSA contains multiple hydrophobic binding sites that can bind with ligands (He & Carter, 1992). In the 1970s, Sudlow et al. (Sudlow, Birkett, & Wade, 1976) found two specific and widely recognized binding sites in HSA by using fluorescence probes. Those two primary sites, I and II, are highly adaptable for binding to several commonly used drugs (Ghuman et al., 2005). Occasionally it has been found in our laboratory that OG also exhibits a similar property in the presence of HSA. This work extensively investigated this property of OG. In addition to HSA, some other commonly used proteins in ligand–macromolecule interaction studies along with three fluorescent amino acids, were used for comparison (Ghosh, Dolai, Patra, & Dey, 2015; Tatlidil, Ucuncu, & Akdogan, 2015; Wu, Zhao, Yang, & Yan, 2015; Zhang et al., 2015). Ligand–macromolecule binding may affect the conformation and stability of the same protein; thus, circular dichroism (CD) study was performed. Nuclear magnetic resonance (NMR) was performed to observe and characterize the interaction between OG and HSA. WATERGATE solvent suppression sequence was used to suppress H2O signal in the samples. Saturation transfer difference (STD) was employed to characterize the OG that
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2.4. CD measurement
Fig. 1. Molecular structure of octyl gallate (OG).
interacts with HSA (Mayer & Meyer, 2001). Displacement and molecular docking techniques were used to establish the binding of OG on the two primary sites in HSA. OG, as a food additive, can be used at limited amounts, e.g., the maximum permitted levels of OG in foods in EU is 25–400 mg/kg. Several analytical methods, including high-performance liquid chromatography, gas chromatography, and capillary electrophoresis, have been developed to measure OG (Li, Chao, Sun, Yang, & Chu, 2009; Perrin & Meyer, 2002; Wang et al., 2012). Fluorescence probes are usually developed to quantitatively detect proteins in biological fluids. In turn, the possibility of using HSA to quantify OG was investigated. The results of this study may provide valuable information that will elucidate the selective and sensitive fluorescent property of OG and may offer a new method for rapid OG quantification. 2. Materials and methods 2.1. Reagents HSA (essentially fatty acid-free), pepsin from porcine gastric mucosa, trypsin from bovine pancreas, deuterium oxide (D2O, 99.9% D atom), and DMSO-d6 (99.9% D atom) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA, fatty acid free) was obtained from EMD Millipore Corp. (Billerica, MA, USA). Different concentrations of HSA and BSA in phosphate buffer saline (PBS; pH = 7.40) were prepared using the extinction coefficients of 35700 M1 cm1 for HSA and 44720 M1 cm1 for BSA at 280 nm (Gelamo, Silva, Imasato, & Tabak, 2002). Egg white lysozyme (LYZ) was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). The concentrations of LYZ, pepsin, and trypsin in PBS buffer were prepared based from their listed molecular weights of 14.4, 35.0, and 23.8 kDa, respectively, and their final concentrations were determined using protein concentration of (1 g/L) = 1.45A280 0.74A260 (Yoshizaki, Oshima, & Imahori, 1971). OG, tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), warfarin (WF), phenylbutazone (PB), and ibuprofen (IP) were purchased from J&K Scientific, Ltd. (Beijing, China). Margarine and edible oil were acquired from a local supermarket. 2.2. Fluorescence spectroscopy study Fluorescence analyses were conducted on a Cary Eclipse fluorescence spectrophotometer (Varian, USA), equipped with a 1 cm quartz cell. Fluorescence spectra were obtained using 5/5 nm (excitation/emission) slit widths at kex = 280 nm and 10/5 nm (excitation/emission) slit widths at kex = 320 nm. 2.3. Fluorescence lifetime measurements Fluorescence lifetime was measured through time-correlated single photon counting by using a FluoroLog-3 modular spectrofluorometer (Horiba, France). The time-resolved quenching of proteins fluorescence by using OG were recorded at excitation and emission wavelengths of 280 and 345 nm, respectively. The HSA/ BSA/LYZ concentration was set at 4.0 lM and that of OG at 4.0 and 8.0 lM.
CD spectra were recorded on a CD spectrometer (Model 400, AVIV, USA). CD was measured at a constant HSA/BSA concentration of 2.0 lM, whereas complex concentration was varied from 0 lM to 8.0 lM [ri = (OG)/(HSA/BSA) = 0:1, 2:1, 4:1]. Spectra were obtained at 298 K in a 2 mm quartz cell from 250 nm to 190 nm with a step size of 1 nm, a band width of 1 nm and an average time of 0.5 s. Each spectrum was scanned in triplicate and averaged for graphing and further analyses. 2.5. NMR study All NMR experiments were performed on a Varian 700 MHz Inova spectrometer, operating at 298 K and running under VNMRJ software (version 2.1B). The stock solutions of OG (40 mM), WF (80 mM), and IP (80 mM) were prepared in DMSO-d6 for further use. The stock solution of HSA was prepared in 0.01 M PBS buffer (pH = 7.40, 50% [v/v] D2O, and H2O mixture). The samples for final screening contained 0.01 mM HSA and 0.4 mM OG. Subsequently, displacement studies were performed by adding WF/IP (0.8 mM) to the previous screening samples. The concentration of DMSO in all of the samples was less than 2% (v/v). Two 1D spectra, namely, 1H WATERGATE and STD, were obtained. The WATERGATE spectrum was obtained within 2.4 min with both the acquisition and relaxation times set to 2 s and 32 scans. STD experiment was performed at the acquisition time of 1 s, 32 dummy scans, relaxation delay of 0.1 s, and a 2 s Gauss pulse train with an alternating irradiation frequency of 0.7 or 45 ppm. The total acquisition time was 15 min with 256 scans. All spectra were processed and analyzed using MestReNova software. 2.6. Molecular modelling The crystal structure of HSA (2BXG) was obtained from the Brookhaven Protein Data Bank for docking simulations. Hybridization states, charges, and angles were subsequently assigned to the protein structure with missing bond orders. Explicit H atoms were added at pH 7.40. To prepare the ligand, the 3D structure of OG was generated using ChemBioOffice 2010, and the structure was optimized using Discovery Studio 3.1 (DS 3.1; Accelrys Co., Ltd., USA), which was provided by the State Key Laboratory of Biotherapy (Sichuan University, China). The CDOCKER docking program implemented in DS 3.1 was used for docking simulation in this study. 2.7. Simulated OG extraction 2 g of margarine and edible oil were weighed into a centrifuge tube, respectively. 10 ml of hexane were added and the mixture was vigorously shaken for 3 min using a vortex mixer. Simulated OG extraction was performed using 5 ml of acetonitrile from the hexane dilutions (Irache, Vega, & Ezpeleta, 1991). The acetonitrile extract was quantitatively transferred into a round-bottomed flask. The procedure was repeated 3 times, and then the extracts were combined. The acetonitrile extract was subsequently evaporated to 2 ml by using a rotary evaporator under a reduced pressure (<100 mbar), at 40 °C (Perrin & Meyer, 2002). 3. Results and discussion 3.1. Sensitive and selective fluorescent property of OG HSA fluorescence originates from the Trp, Tyr, and Phe residues, whereas HSA emission is primarily attributed to the Trp-214 resi-
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due. The Phe residue produces a considerably low quantum yield, and the energy absorbed by Phe and Tyr is often transferred to Trp residues in the same protein (Lakowicz, 2009, chap. 13). The effect of OG on the fluorescence intensity of HSA (4 lM) is shown in Fig. 2, in which HSA emits an evident fluorescence emission band at 338 nm (kex = 280 nm). The fluorescence of HSA decreased obviously with the addition of OG (0–6 lM). OG (6 lM) alone nearly displayed no fluorescence, although its fluorescence was obviously enhanced (kem 365 nm) in the presence of HSA. This enhancement was more remarkable at kex = 320 nm (inset in Fig. 2), in which the proteins were not excited. OG (4 lM) yielded approximately 49-fold increment in emission intensity (Fig. 3). However, this phenomenon cannot arbitrarily occur in all interactions. The fluorescence of OG was no longer enhanced when HSA was replaced by Trp/Tyr/Phe solution or some other commonly used proteins, except BSA. OG (4 lM) yielded an approximately 11-fold increment in the presence of BSA (4 lM). It was hypothesized that the specific structure of HSA provided the basic conditions for enhancement of OG fluorescence. BSA displays approximately 76% sequence homology with HSA (Majorek et al., 2012). It also had the specific structure for enhancement of OG fluorescence. But the structure in BSA still had a little different with that in HSA as OG displayed obvious fluorescence enhancement
efficiency difference in BSA and HSA. At kex = 280 nm, it was found that adding OG slightly reduced the fluorescence of HSA/BSA but irregularly quenched LYZ (Fig. S1). The fluorescence quenching observed is related to the fluorescence enhancement of OG. Fluorescence quenching refers to any process that reduces the fluorescence intensity of a sample. A variety of molecular interactions, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching, can result in fluorescence quenching. The selective reduction in intensity of HSA/BSA and the increase in OG fluorescence phenomenon are consistent with energy transfer phenomenon, i.e., Förster resonance energy transfer (FRET) phenomenon (Chakraborty, Das, & Halder, 2011; Förster, 1948). In addition, OG could still be excited at kex = 320 nm, at which HSA/BSA could not be excited. FRET physically originates from the weak electromagnetic coupling of two dipoles (Yun et al., 2005). It was considered that this electromagnetic coupling did not require a resonant electronic transition, as well as surface energy transfer (Sen, Sadhu, & Patra, 2007). In fact, Förster once proposed that excitation transfer is by no means the only possible mechanism of energy transfer (Förster, 1960). Along with energy transfer, the lifetime of a donor will decrease. The fluorescence lifetime of HSA/BSA/LYZ was then determined (kex = 280 nm and kem = 345 nm) in the absence and presence of OG (inset in Fig. 3). The data obtained were analyzed using the tail-fitting method, and the quality of each fit was assessed by v2 values and residuals. The amplitude-weighted lifetime (‹s›) for biexponential iterative fitting was calculated from the decay times and pre-exponential factors (a) by using the following equation (Ray, Badugu, & Lakowicz, 2015):
< s >¼ a1 s1 þ a2 s2 þ a3 s3
Fig. 2. Fluorescence spectra of OG–HSA interaction at kex = 280 nm and kex = 320 nm (inset). C(HSA) = 4 lM, C(OG) = 0, 1, 2, 3, 4, 5, and 6 lM, respectively.
Fig. 3. Relative fluorescence intensity of OG (4 lM) with three fluorescent amino acids and different proteins (4 lM). The inset was the fluorescence life time of HSA/BSA/LYZ in the absence and presence of OG (kex = 280 nm and kem = 345 nm). C(HSA/BSA/LYZ) = 4 lM, C(OG) = 0 lM (black), 4 lM (red), 8 lM (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ð1Þ
The amplitude-weighted lifetime was chosen as an important parameter to explore the behaviour of OG bound to the three typical proteins. In the absence of OG, the amplitude-weighted lifetimes of HSA, BSA, and LYZ were 5.348, 6.230, and 2.002 ns, respectively (Table S1). In the presence of OG, the decay time of HSA considerably decreased corresponding to the obviously enhanced OG fluorescence. A similar association was observed in BSA and LYZ. In BSA, the moderate reduction in decay time corresponded to the moderate fluorescence enhancement of OG. OG did not reduce the decay time of LYZ, and OG fluorescence remained unchanged in LYZ. Collisional quenching can also result in lifetime reduction, but can be excluded in this study because the fluorescence enhancement of OG requires a complex process (Section 3.2). Energy transfer can be characterized based on the efficiency of the transfer. Transfer efficiencies (E) were calculated from the lifetimes of the donor in the absence (‹s0›) and presence (‹s›) of acceptor: E = 1 ‹s›/‹s0›. The lifetime of a micromolecule is usually less than that of macromolecules. Thus, the lifetime of OG was not considered. The corresponding E at a molar ratio of 1:1 were 27.0% for HSA and 6.6% for BSA. E is positively correlated with the efficiency of OG fluorescence enhancement. Thus, FRET definitely occurred during OG–HSA/BSA interaction. CD spectroscopy plays an important role in studying protein folding because this technique allows the characterization of secondary and tertiary structures of proteins in their native, unfolded, and partially folded states. Although an energy transfer obviously occurred in OG–HSA/BSA interaction, OG exerted minimal effects on the structures of HSA and BSA (Fig. S2). HSA and BSA exhibited two negative ellipticities at 208 and 220 nm, which are characteristic of a-helix structures in proteins. The shapes of the CD spectra with and without OG were similar. The negative bands did not shift, and their intensity remained nearly constant when system errors are considered. Thus, the fluorescence enhancement that occurred in OG–HSA/BSA interaction was not caused by structural
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changes in the proteins. This interesting phenomenon in OG–HSA interaction was then targeted for investigation. 3.2. Insight into OG binding to HSA Interactions between biological macromolecules and ligands can be possibly observed and characterized using NMR methods (Meyer & Peters, 2003). STD NMR, which can deliver information in the form of ligand signals in the NMR spectrum, is commonly used (Mudgal, Keresztes, Feigenson, & Rizvi, 2016). In this method, selective irradiation of the protein NMR spectrum results in saturation of the protein signals and of any ligand protons interacting with the protein. Subtraction of this spectrum from the one collected in non-saturated protein and ligand conditions gives the saturation transfer difference spectrum, in which only proteininteracting protons are visible and the epitope is mapped by analysing the signal intensities of these protons in the STD spectrum (Nienhaus, 2005, chap. 10; Wagstaff, Taylor, & Howard, 2013). OG displayed seven kinds of proton signals in H2O—D2O mixture (Fig. S3). The benzene ring signal at 6.9 ppm was atypical. The appearance of OG signals in STD spectrum indicated that OG interacted with HSA, that is, OG formed a complex with HSA. WF and IP specifically bind to Sudlow sites I and II, respectively. Addition of WF/IP and observation of the intensity of the STD signal of the OG can allow differentiation of binding to the same or to a second binding site. After determining their binding site in HSA, they were chosen as displacement probes. Addition of WF did not influence the STD signal of OG (Fig. 4d). The STD signals of WF and OG were all clearly visible at 6.9–8 ppm. However, adding IP to the HSA/OG mixture seemed to completely suppress the STD signal intensity of OG (Fig. 4e). Only the STD signals of IP were clearly visible. A competition for the same binding site caused IP to displace OG, and we could preliminary conclude that OG mainly bound to the IP site, i.e., Sudlow site II. This conclusion was further confirmed using the method of Sudlow et al. (Sudlow, Birkett, & Wade, 1976). WF was replaced by IP because the fluorescence of WF was also enhanced in the presence of HSA and BSA (kex = 320 nm; Fig. S4). PB did not inhibit the fluorescence enhancement of OG, whereas IP significantly did (Fig. 4f). OG mainly bound to Sudlow site II was further confirmed. When OG was freed from HSA and BSA, the fluorescence enhancement decreased rapidly, indicating that the formation of a stable complex was an important prerequisite for efficient fluorescence enhancement. The binding modelling was then established using the CDOCKER docking program, a molecular dynamic simulatedannealing-based algorithm implemented in DS 3.1 (Wu, Robertson, Brooks, & Vieth, 2003). OG was docked with the Sudlow site II in 2BXG. The detail docking parameters and processes used were similar to those in our previous work (Wang, Ma, He, Li, & Li, 2015). The pose with the lowest CDOCKER energy was chosen as the most suitable pose for the subsequent pose analysis. Fig. 5 shows that OG inserted well into the hydrophobic cavity of Sudlow site II. OG formed four hydrogen bonds because of its strong donor and acceptor groups. H-bonding during OG–HSA complex formation can effectively balance the adverse energetic effects of water displacement. p–cation interaction occurs between a positively charged molecule and a p system. In the OG–HSA interaction, Arg-410 acted as cation participating in the p–cation interaction. Those interactions were necessary for the formation of a stable OG–HSA complex. Dey et al. found that when the OH group was replaced by the methoxy group, the fluorescence of their probes showed very weak enhancement. Strong H-bonding was therefore necessary in optical signaling (Dey, Gaur, Giri, & Ghosh, 2016). The location of OG in HSA was relatively far from the Trp-214 residue, which is primarily attributed to the HSA emission. OG quenched HSA from a long distance. Both static quenching (ground-state
Fig. 4. The 1H (a) STD spectrum for free OG, (b) WATERGATE and (c) STD spectrum for the OG-HSA interaction and effect of (d) WF/(e)IP on the STD spectrum of the OG-HSA interaction at 298 K (pH = 7.40). The on-resonance irradiation was performed at a chemical shift of 0.7 ppm, whereas the off-resonance irradiation was conducted at 45 ppm. C(HSA) = 0.01 mM, C(OG) = 0.4 mM and C(WF/IP) = 0.8 mM. (f) Effect of PB/IP on the fluorescence of OG–HSA interaction (kex = 320 nm). C(HSA) = C(OB) = C(PB/IP) = 4 lM.
complex formation) and dynamic quenching require molecular contact between the fluorophore and quencher (Lakowicz, 2009). FRET results from long-range dipole–dipole interaction between a donor and an acceptor molecule. Thus, FRET was further confirmed to cause the interesting phenomenon in OG–HSA interaction. 3.3. Possibility for quantitative OG recognition Fluorescence-based assays offer widespread applications in the fluorescence imaging of various analytes owing to their rapid, nondestructive, selective, and sensitive measurement of emission signals (Guo, Park, Yoon, & Shin, 2014). Foods added with antioxidants include fats and oils, dairy produce, cereals, snacks, and food flavourings. The possibility for quantitative OG recognition in margarine and edible oil by using HSA was then investigated. OG has not been officially approved as a food additive in China. The acetonitrile extracts of margarine (sample A) and edible oil (sample B) used in this study did not contain OG and nearly showed no fluorescence at kex = 280, 320 nm (Fig. S5). The presence of sample A/B (4‰, v/v) did not affect the intrinsic fluorescence of HSA (4 lM). By contrast, the fluorescence enhancement of
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Fig. 5. Molecular modelling of OG docked to Sudlow site II of HSA.
Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the National Development and Reform Commission and Education of China (Grant No. 2014BW011). Thanks to School of Life Science, University of Science and Technology of China for NMR technical assistance. Appendix A. Supplementary data
Fig. 6. Relative fluorescence intensity of OG (0.14–1.69 mg/ml) in HSA (4 lM) when in the presence of 4‰ (v/v) acetonitrile extract of margarine (j, R2 = 0.983) or edible oil (d, R2 = 0.991).
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2016. 09.157. References
OG (4 lM) was inhibited at different degrees (kex = 320 nm). The inhibition ability of sample B was much stronger than that of sample A. The samples (4‰, v/v) mixed with HSA (4 lM) were spiked with different OG concentrations. A good linear correlation between the relative fluorescence enhancement of OG and its amount was observed (Fig. 6). HSA can be applied to determine OG concentration. However, foods contain a considerably low amount of OG. The use of standard addition method is more appropriate to better quantify OG. Many ligands can bind to HSA as it contains multiple hydrophobic binding sites (Vaya, LhiaubetVallet, Jimenez, & Miranda, 2014). Other non-fluorescent components existing in samples A and B more or less interfered with the fluorescence enhancement of OG. Those components may have hindered the basic complexation or the electromagnetic coupling. Hence, the standard curve for quantitative recognition of OG should be built in corresponding blank samples.
4. Conclusions In summary, the selective and sensitive fluorescent property of OG was extensively analyzed. The fluorescence of OG was obviously enhanced in the presence of HSA. After performing multiple investigations and analyses, it can be concluded that this phenomenon was a FRET phenomenon. The formation of a stable complex was an important prerequisite for efficient fluorescence enhancement. A special structure of donor, such as HSA, was also a prerequisite in fluorescence enhancement. The selective and sensitive fluorescent property of OG can be used to determine OG concentration through standard addition method, which must be performed under certain conditions.
Anees, P., Sreejith, S., & Ajayaghosh, A. (2014). Self-assembled near-infrared dye nanoparticles as a selective protein sensor by activation of a dormant fluorophore. Journal of the American Chemical Society, 136(38), 13233–13239. Aruoma, O. I., Murcia, A., Butler, J., & Halliwell, B. (1993). Evaluation of the antioxidant and prooxidant actions of gallic acid and its derivatives. Journal of the American Chemical Society, 41(11), 1880–1885. Chakraborty, J., Das, N., & Halder, U. C. (2011). Unfolding diminishes fluorescence resonance energy transfer (FRET) of lysine modified beta-lactoglobulin: Relevance towards anti-HIV binding. Journal of Photochemistry and Photobiology B: Biology, 102(1), 1–10. Dey, G., Gaur, P., Giri, R., & Ghosh, S. (2016). Optical signaling in biofluids: a nondenaturing photostable molecular probe for serum albumins. Chemical Communications, 52(9), 1887–1890. Efsa, ANS Panel (EFSA Panel on Food Additives and Nutrient Sources Added to Food) (2015). Scientific Opinion on the re-evaluation of octyl gallate (E 311) as a food additive. European Food Safety Authority Journal, 13(10), 4248. Förster, T. (1948). Zwischenmolekulare energiewanderung und fluoreszenz. Annalen der Physik Berlin, 437(1–2), 55–75. Förster, T. (1960). Transfer mechanisms of electronic excitation energy. Radiation Research Supplements, 326–339. Gelamo, E. L., Silva, C. H. T. P., Imasato, H., & Tabak, M. (2002). Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling. BBA-Protein Structure, 1594(1), 84–99. Ghosh, S., Dolai, S., Patra, T., & Dey, J. (2015). Solution behavior and interaction of pepsin with carnitine based cationic surfactant: fluorescence, circular dichroism, and calorimetric studies. The Journal of Physical Chemistry B, 119 (39), 12632–12643. Ghuman, J., Zunszain, P. A., Petitpas, I., Bhattacharya, A. A., Otagiri, M., & Curry, S. (2005). Structural basis of the drug-binding specificity of human serum albumin. Journal of Molecular Biology, 353(1), 38–52. Guo, Z. Q., Park, S., Yoon, J. Y., & Shin, I. (2014). Recent progress in the development of near-infrared fluorescent probes for bioimaging applications. Chemical Society Reviews, 43(1), 16–29. He, X. M., & Carter, D. C. (1992). Atomic structure and chemistry of human serum albumin. Nature, 358(16), 209–215. He, X. P., Zang, Y., James, T. D., Li, J., & Chen, G. R. (2015). Probing disease-related proteins with fluorogenic composite materials. Chemical Society Reviews, 44, 4239–4248. Irache, J. M., Vega, F. A., & Ezpeleta, I. (1991). Antioxidants in some pharmaceuticals, cosmetics and food from the European market. Pharmaceutica Acta Helvetiae, 67 (5–6), 152–155.
Q. Wang et al. / Food Chemistry 219 (2017) 268–273 Joint FAO/WHO Expert Committee on Food Additives (1962). Evaluation of the toxicity of a number of antimicrobials and antioxidants. World Health Organization Technical Report Series, 228, 61–65. Lakowicz, J. R. (2009). Principles of fluorescence spectroscopy (3rd ed.). Singapore: Springer. Li, X. Q., Chao, J., Sun, Y. Y., Yang, M. L., & Chu, X. G. (2009). Analysis of synthetic antioxidants and preservatives in edible vegetable oil by HPLC/TOF-MS. Food Chemistry, 113(2), 692–700. Liu, J., Chen, Y., Wang, W., Feng, J., Liang, M., Ma, S., & Chen, X. (2016). ‘‘Switch-On” fluorescent sensing of ascorbic acid in food samples based on carbon quantum dots-MnO2 probe. Journal of Agriculture and Food Chemistry, 64(1), 371–380. Majorek, K. A., Porebski, P. J., Dayal, A., Zimmerman, M. D., Jablonska, K., Stewart, A. J., ... Minor, W. (2012). Structural and immunologic characterization of bovine, horse, and rabbit serum albumins. Molecular Immunology, 52(3), 174–182. Mayer, M., & Meyer, B. (2001). Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. Journal of the American Chemical Society, 123(25), 6108–6117. Meyer, B., & Peters, T. (2003). NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angewandte Chemie International Edition, 42(8), 864–890. Mudgal, S., Keresztes, I., Feigenson, G. W., & Rizvi, S. S. (2016). Controlling the taste receptor accessible structure of rebaudioside A via binding to bovine serum albumin. Food Chemistry, 197, 84–91. Nienhaus, G. U. (2005). Protein-ligand interactions. USA: Springer. Perrin, C., & Meyer, L. (2002). Quantification of synthetic phenolic antioxidants in dry foods by reversed-phase HPLC with photodiode array detection. Food Chemistry, 77(1), 93–100. Ray, K., Badugu, R., & Lakowicz, J. R. (2015). Bloch surface wave-coupled emission from quantum dots by ensemble and single molecule spectroscopy. RSC Advance, 5(67), 54403–54411. Sen, T., Sadhu, S., & Patra, A. (2007). Surface energy transfer from rhodamine 6G to gold nanoparticles: A spectroscopic ruler. Applied Physics Letters, 91(4), 043104. Shahidi, F., Janitha, P. K., & Wanasundara, P. D. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition, 32(1), 67–103. Sudlow, G., Birkett, D. J., & Wade, D. N. (1976). Further characterization of specific drug binding sites on human serum albumin. Molecular Pharmacology, 12(6), 1052–1061.
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Tatlidil, D., Ucuncu, M., & Akdogan, Y. (2015). Physiological concentrations of albumin favor drug binding. Physical Chemistry Chemical Physics, 17(35), 22678–22685. Vaya, I., Lhiaubet-Vallet, V., Jimenez, M. C., & Miranda, M. A. (2014). Photoactive assemblies of organic compounds and biomolecules: Drug-protein supramolecular systems. Chemical Society Reviews, 43(12), 4102–4122. Wagstaff, J. L., Taylor, S. L., & Howard, M. J. (2013). Recent developments and applications of saturation transfer difference nuclear magnetic resonance (STD NMR) spectroscopy. Molecular BioSystems, 9(4), 571–577. Wang, Q., Ma, X. L., He, J. W., Li, Y. Z., & Li, H. (2015). Insights into the fatty acid ester norethisterone enanthate binding to human albumin: fluorescence, circular dichroism, and docking investigations. RSC Advance, 5, 44696–44704. Wang, J. Y., Wu, H. L., Chen, Y., Sun, Y. M., Yu, Y. J., Zhang, X. H., & Yu, R. Q. (2012). Fast analysis of synthetic antioxidants in edible vegetable oil using trilinear component modeling of liquid chromatography–diode array detection data. Journal of Chromatography A, 1264, 63–71. Wu, G., Robertson, D. H., Brooks, C. L., & Vieth, M. (2003). Detailed analysis of grid-based molecular docking: A case study of CDOCKER—A CHARMm-based MD docking algorithm. Journal of Computational Chemistry, 24(13), 1549–1562. Wu, L., Zhao, W., Yang, R., & Yan, W. (2015). Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system. Food Chemistry, 175, 115–120. Yoshizaki, F., Oshima, T., & Imahori, K. (1971). Studies on phosphoglucomutase from an extreme thermophile, flavobacterium thermophilum HB8. Journal of Biochemistry, 69(6), 1083–1089. Yun, C. S., Javier, A., Jennings, T., Fisher, M., Hira, S., Peterson, S., ... Strouse, G. F. (2005). Nanometal surface energy transfer in optical rulers, breaking the FRET barrier. Journal of the American Chemical Society, 127(9), 3115–3119. Zhang, R., Sun, T., Liu, C. G., Song, W., Cao, Z. Z., & Liu, R. T. (2015). New insights into the toxicity of n-butanol to trypsin: Spectroscopic and molecular docking descriptions. Journal of Biochemical and Molecular Toxicology, 29(9), 418–425. Zhu, T., Du, J. J., Cao, W. B., Fan, J. L., & Peng, X. J. (2016). Microenvironment-sensitive fluorescent dyes for recognition of serum albumin in urine and imaging in living cells. Industrial and Engineering Chemistry Research, 55(3), 527–533.
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Octyl gallate: An antioxidant demonstrating selective and sensitive fluorescent property Qing Wang, Yongkui Zhang, Hui Li ⇑ College of Chemical Engineering, Sichuan University, Chengdu 610065, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 7 June 2016 Received in revised form 16 August 2016 Accepted 26 September 2016 Available online 28 September 2016 Chemical compounds studied in this article: PubChem CID: 61253 Keywords: Octyl gallate Fluorescence enhancement Förster resonance energy transfer (FRET) Complex formation Displacement
a b s t r a c t Octyl gallate (OG) is an internationally recognized antioxidant that demonstrates selective and sensitive fluorescent property. The fluorescence of OG can be selectively enhanced in the presence of human serum albumin (HSA) and bovine serum albumin (BSA). The specific structures of HSA and BSA provided the basic conditions for fluorescence enhancement. OG yielded approximately 49- and 11-fold increments in emission intensity in the presence of HSA and BSA at a molar ratio of 1:1, respectively. The lifetimes of HSA and BSA correspondingly decreased. A Förster resonance energy transfer phenomenon occurred during interaction between OG and HSA or BSA. Our in-depth investigation of OG–HSA interaction showed that formation of a stable complex was an important prerequisite to efficiently enhance the fluorescence of OG. The selective and sensitive fluorescent property of OG can possibly be used to determine OG concentration via the standard addition method, which must be performed under certain conditions. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Autoxidation naturally occurs between molecular oxygen and unsaturated lipids in the environment (Shahidi, Janitha, & Wanasundara, 1992). Deterioration of foodstuffs is often prevented by adding lipid soluble antioxidants during food processing (Aruoma, Murcia, Butler, & Halliwell, 1993). Octyl gallate (OG, Fig. 1) is an internationally recognized antioxidant that can be added into foods to inhibit lipid autoxidation (Joint FAO/WHO Expert Committee on Food Additives, 1962). OG has long been evaluated by the Scientific Committee on Food and the Joint FAO/WHO Expert Committee on Food Additives; in addition, OG has been approved in many countries and regions, such as in European Union (EU) and Australia, as a food additive (EFSA ANS Panel, 2015). Fluorescence-based assays involving small molecules that can induce a strong optical signal while interacting with proteins have considerably attracted the interest of researchers (Dey, Gaur, Giri, & Ghosh, 2016; Liu et al., 2016). These probes themselves usually emit no or weak fluorescence, although the fluorescence of these probes can be significantly enhanced when they interact with proteins. Many research groups have developed highly efficient ⇑ Corresponding author. E-mail address: [email protected] (H. Li). http://dx.doi.org/10.1016/j.foodchem.2016.09.157 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
molecular probes to detect proteins, such as human serum albumin (HSA) (Anees, Sreejith, & Ajayaghosh, 2014; He, Zang, James, Li, & Chen, 2015; Zhu, Du, Cao, Fan, & Peng, 2016). Interestingly, fluorescence enhancement does not occur in a specific site in HSA. Crystallographic analyses have revealed that HSA contains multiple hydrophobic binding sites that can bind with ligands (He & Carter, 1992). In the 1970s, Sudlow et al. (Sudlow, Birkett, & Wade, 1976) found two specific and widely recognized binding sites in HSA by using fluorescence probes. Those two primary sites, I and II, are highly adaptable for binding to several commonly used drugs (Ghuman et al., 2005). Occasionally it has been found in our laboratory that OG also exhibits a similar property in the presence of HSA. This work extensively investigated this property of OG. In addition to HSA, some other commonly used proteins in ligand–macromolecule interaction studies along with three fluorescent amino acids, were used for comparison (Ghosh, Dolai, Patra, & Dey, 2015; Tatlidil, Ucuncu, & Akdogan, 2015; Wu, Zhao, Yang, & Yan, 2015; Zhang et al., 2015). Ligand–macromolecule binding may affect the conformation and stability of the same protein; thus, circular dichroism (CD) study was performed. Nuclear magnetic resonance (NMR) was performed to observe and characterize the interaction between OG and HSA. WATERGATE solvent suppression sequence was used to suppress H2O signal in the samples. Saturation transfer difference (STD) was employed to characterize the OG that
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2.4. CD measurement
Fig. 1. Molecular structure of octyl gallate (OG).
interacts with HSA (Mayer & Meyer, 2001). Displacement and molecular docking techniques were used to establish the binding of OG on the two primary sites in HSA. OG, as a food additive, can be used at limited amounts, e.g., the maximum permitted levels of OG in foods in EU is 25–400 mg/kg. Several analytical methods, including high-performance liquid chromatography, gas chromatography, and capillary electrophoresis, have been developed to measure OG (Li, Chao, Sun, Yang, & Chu, 2009; Perrin & Meyer, 2002; Wang et al., 2012). Fluorescence probes are usually developed to quantitatively detect proteins in biological fluids. In turn, the possibility of using HSA to quantify OG was investigated. The results of this study may provide valuable information that will elucidate the selective and sensitive fluorescent property of OG and may offer a new method for rapid OG quantification. 2. Materials and methods 2.1. Reagents HSA (essentially fatty acid-free), pepsin from porcine gastric mucosa, trypsin from bovine pancreas, deuterium oxide (D2O, 99.9% D atom), and DMSO-d6 (99.9% D atom) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA, fatty acid free) was obtained from EMD Millipore Corp. (Billerica, MA, USA). Different concentrations of HSA and BSA in phosphate buffer saline (PBS; pH = 7.40) were prepared using the extinction coefficients of 35700 M1 cm1 for HSA and 44720 M1 cm1 for BSA at 280 nm (Gelamo, Silva, Imasato, & Tabak, 2002). Egg white lysozyme (LYZ) was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). The concentrations of LYZ, pepsin, and trypsin in PBS buffer were prepared based from their listed molecular weights of 14.4, 35.0, and 23.8 kDa, respectively, and their final concentrations were determined using protein concentration of (1 g/L) = 1.45A280 0.74A260 (Yoshizaki, Oshima, & Imahori, 1971). OG, tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), warfarin (WF), phenylbutazone (PB), and ibuprofen (IP) were purchased from J&K Scientific, Ltd. (Beijing, China). Margarine and edible oil were acquired from a local supermarket. 2.2. Fluorescence spectroscopy study Fluorescence analyses were conducted on a Cary Eclipse fluorescence spectrophotometer (Varian, USA), equipped with a 1 cm quartz cell. Fluorescence spectra were obtained using 5/5 nm (excitation/emission) slit widths at kex = 280 nm and 10/5 nm (excitation/emission) slit widths at kex = 320 nm. 2.3. Fluorescence lifetime measurements Fluorescence lifetime was measured through time-correlated single photon counting by using a FluoroLog-3 modular spectrofluorometer (Horiba, France). The time-resolved quenching of proteins fluorescence by using OG were recorded at excitation and emission wavelengths of 280 and 345 nm, respectively. The HSA/ BSA/LYZ concentration was set at 4.0 lM and that of OG at 4.0 and 8.0 lM.
CD spectra were recorded on a CD spectrometer (Model 400, AVIV, USA). CD was measured at a constant HSA/BSA concentration of 2.0 lM, whereas complex concentration was varied from 0 lM to 8.0 lM [ri = (OG)/(HSA/BSA) = 0:1, 2:1, 4:1]. Spectra were obtained at 298 K in a 2 mm quartz cell from 250 nm to 190 nm with a step size of 1 nm, a band width of 1 nm and an average time of 0.5 s. Each spectrum was scanned in triplicate and averaged for graphing and further analyses. 2.5. NMR study All NMR experiments were performed on a Varian 700 MHz Inova spectrometer, operating at 298 K and running under VNMRJ software (version 2.1B). The stock solutions of OG (40 mM), WF (80 mM), and IP (80 mM) were prepared in DMSO-d6 for further use. The stock solution of HSA was prepared in 0.01 M PBS buffer (pH = 7.40, 50% [v/v] D2O, and H2O mixture). The samples for final screening contained 0.01 mM HSA and 0.4 mM OG. Subsequently, displacement studies were performed by adding WF/IP (0.8 mM) to the previous screening samples. The concentration of DMSO in all of the samples was less than 2% (v/v). Two 1D spectra, namely, 1H WATERGATE and STD, were obtained. The WATERGATE spectrum was obtained within 2.4 min with both the acquisition and relaxation times set to 2 s and 32 scans. STD experiment was performed at the acquisition time of 1 s, 32 dummy scans, relaxation delay of 0.1 s, and a 2 s Gauss pulse train with an alternating irradiation frequency of 0.7 or 45 ppm. The total acquisition time was 15 min with 256 scans. All spectra were processed and analyzed using MestReNova software. 2.6. Molecular modelling The crystal structure of HSA (2BXG) was obtained from the Brookhaven Protein Data Bank for docking simulations. Hybridization states, charges, and angles were subsequently assigned to the protein structure with missing bond orders. Explicit H atoms were added at pH 7.40. To prepare the ligand, the 3D structure of OG was generated using ChemBioOffice 2010, and the structure was optimized using Discovery Studio 3.1 (DS 3.1; Accelrys Co., Ltd., USA), which was provided by the State Key Laboratory of Biotherapy (Sichuan University, China). The CDOCKER docking program implemented in DS 3.1 was used for docking simulation in this study. 2.7. Simulated OG extraction 2 g of margarine and edible oil were weighed into a centrifuge tube, respectively. 10 ml of hexane were added and the mixture was vigorously shaken for 3 min using a vortex mixer. Simulated OG extraction was performed using 5 ml of acetonitrile from the hexane dilutions (Irache, Vega, & Ezpeleta, 1991). The acetonitrile extract was quantitatively transferred into a round-bottomed flask. The procedure was repeated 3 times, and then the extracts were combined. The acetonitrile extract was subsequently evaporated to 2 ml by using a rotary evaporator under a reduced pressure (<100 mbar), at 40 °C (Perrin & Meyer, 2002). 3. Results and discussion 3.1. Sensitive and selective fluorescent property of OG HSA fluorescence originates from the Trp, Tyr, and Phe residues, whereas HSA emission is primarily attributed to the Trp-214 resi-
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due. The Phe residue produces a considerably low quantum yield, and the energy absorbed by Phe and Tyr is often transferred to Trp residues in the same protein (Lakowicz, 2009, chap. 13). The effect of OG on the fluorescence intensity of HSA (4 lM) is shown in Fig. 2, in which HSA emits an evident fluorescence emission band at 338 nm (kex = 280 nm). The fluorescence of HSA decreased obviously with the addition of OG (0–6 lM). OG (6 lM) alone nearly displayed no fluorescence, although its fluorescence was obviously enhanced (kem 365 nm) in the presence of HSA. This enhancement was more remarkable at kex = 320 nm (inset in Fig. 2), in which the proteins were not excited. OG (4 lM) yielded approximately 49-fold increment in emission intensity (Fig. 3). However, this phenomenon cannot arbitrarily occur in all interactions. The fluorescence of OG was no longer enhanced when HSA was replaced by Trp/Tyr/Phe solution or some other commonly used proteins, except BSA. OG (4 lM) yielded an approximately 11-fold increment in the presence of BSA (4 lM). It was hypothesized that the specific structure of HSA provided the basic conditions for enhancement of OG fluorescence. BSA displays approximately 76% sequence homology with HSA (Majorek et al., 2012). It also had the specific structure for enhancement of OG fluorescence. But the structure in BSA still had a little different with that in HSA as OG displayed obvious fluorescence enhancement
efficiency difference in BSA and HSA. At kex = 280 nm, it was found that adding OG slightly reduced the fluorescence of HSA/BSA but irregularly quenched LYZ (Fig. S1). The fluorescence quenching observed is related to the fluorescence enhancement of OG. Fluorescence quenching refers to any process that reduces the fluorescence intensity of a sample. A variety of molecular interactions, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching, can result in fluorescence quenching. The selective reduction in intensity of HSA/BSA and the increase in OG fluorescence phenomenon are consistent with energy transfer phenomenon, i.e., Förster resonance energy transfer (FRET) phenomenon (Chakraborty, Das, & Halder, 2011; Förster, 1948). In addition, OG could still be excited at kex = 320 nm, at which HSA/BSA could not be excited. FRET physically originates from the weak electromagnetic coupling of two dipoles (Yun et al., 2005). It was considered that this electromagnetic coupling did not require a resonant electronic transition, as well as surface energy transfer (Sen, Sadhu, & Patra, 2007). In fact, Förster once proposed that excitation transfer is by no means the only possible mechanism of energy transfer (Förster, 1960). Along with energy transfer, the lifetime of a donor will decrease. The fluorescence lifetime of HSA/BSA/LYZ was then determined (kex = 280 nm and kem = 345 nm) in the absence and presence of OG (inset in Fig. 3). The data obtained were analyzed using the tail-fitting method, and the quality of each fit was assessed by v2 values and residuals. The amplitude-weighted lifetime (‹s›) for biexponential iterative fitting was calculated from the decay times and pre-exponential factors (a) by using the following equation (Ray, Badugu, & Lakowicz, 2015):
< s >¼ a1 s1 þ a2 s2 þ a3 s3
Fig. 2. Fluorescence spectra of OG–HSA interaction at kex = 280 nm and kex = 320 nm (inset). C(HSA) = 4 lM, C(OG) = 0, 1, 2, 3, 4, 5, and 6 lM, respectively.
Fig. 3. Relative fluorescence intensity of OG (4 lM) with three fluorescent amino acids and different proteins (4 lM). The inset was the fluorescence life time of HSA/BSA/LYZ in the absence and presence of OG (kex = 280 nm and kem = 345 nm). C(HSA/BSA/LYZ) = 4 lM, C(OG) = 0 lM (black), 4 lM (red), 8 lM (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ð1Þ
The amplitude-weighted lifetime was chosen as an important parameter to explore the behaviour of OG bound to the three typical proteins. In the absence of OG, the amplitude-weighted lifetimes of HSA, BSA, and LYZ were 5.348, 6.230, and 2.002 ns, respectively (Table S1). In the presence of OG, the decay time of HSA considerably decreased corresponding to the obviously enhanced OG fluorescence. A similar association was observed in BSA and LYZ. In BSA, the moderate reduction in decay time corresponded to the moderate fluorescence enhancement of OG. OG did not reduce the decay time of LYZ, and OG fluorescence remained unchanged in LYZ. Collisional quenching can also result in lifetime reduction, but can be excluded in this study because the fluorescence enhancement of OG requires a complex process (Section 3.2). Energy transfer can be characterized based on the efficiency of the transfer. Transfer efficiencies (E) were calculated from the lifetimes of the donor in the absence (‹s0›) and presence (‹s›) of acceptor: E = 1 ‹s›/‹s0›. The lifetime of a micromolecule is usually less than that of macromolecules. Thus, the lifetime of OG was not considered. The corresponding E at a molar ratio of 1:1 were 27.0% for HSA and 6.6% for BSA. E is positively correlated with the efficiency of OG fluorescence enhancement. Thus, FRET definitely occurred during OG–HSA/BSA interaction. CD spectroscopy plays an important role in studying protein folding because this technique allows the characterization of secondary and tertiary structures of proteins in their native, unfolded, and partially folded states. Although an energy transfer obviously occurred in OG–HSA/BSA interaction, OG exerted minimal effects on the structures of HSA and BSA (Fig. S2). HSA and BSA exhibited two negative ellipticities at 208 and 220 nm, which are characteristic of a-helix structures in proteins. The shapes of the CD spectra with and without OG were similar. The negative bands did not shift, and their intensity remained nearly constant when system errors are considered. Thus, the fluorescence enhancement that occurred in OG–HSA/BSA interaction was not caused by structural
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changes in the proteins. This interesting phenomenon in OG–HSA interaction was then targeted for investigation. 3.2. Insight into OG binding to HSA Interactions between biological macromolecules and ligands can be possibly observed and characterized using NMR methods (Meyer & Peters, 2003). STD NMR, which can deliver information in the form of ligand signals in the NMR spectrum, is commonly used (Mudgal, Keresztes, Feigenson, & Rizvi, 2016). In this method, selective irradiation of the protein NMR spectrum results in saturation of the protein signals and of any ligand protons interacting with the protein. Subtraction of this spectrum from the one collected in non-saturated protein and ligand conditions gives the saturation transfer difference spectrum, in which only proteininteracting protons are visible and the epitope is mapped by analysing the signal intensities of these protons in the STD spectrum (Nienhaus, 2005, chap. 10; Wagstaff, Taylor, & Howard, 2013). OG displayed seven kinds of proton signals in H2O—D2O mixture (Fig. S3). The benzene ring signal at 6.9 ppm was atypical. The appearance of OG signals in STD spectrum indicated that OG interacted with HSA, that is, OG formed a complex with HSA. WF and IP specifically bind to Sudlow sites I and II, respectively. Addition of WF/IP and observation of the intensity of the STD signal of the OG can allow differentiation of binding to the same or to a second binding site. After determining their binding site in HSA, they were chosen as displacement probes. Addition of WF did not influence the STD signal of OG (Fig. 4d). The STD signals of WF and OG were all clearly visible at 6.9–8 ppm. However, adding IP to the HSA/OG mixture seemed to completely suppress the STD signal intensity of OG (Fig. 4e). Only the STD signals of IP were clearly visible. A competition for the same binding site caused IP to displace OG, and we could preliminary conclude that OG mainly bound to the IP site, i.e., Sudlow site II. This conclusion was further confirmed using the method of Sudlow et al. (Sudlow, Birkett, & Wade, 1976). WF was replaced by IP because the fluorescence of WF was also enhanced in the presence of HSA and BSA (kex = 320 nm; Fig. S4). PB did not inhibit the fluorescence enhancement of OG, whereas IP significantly did (Fig. 4f). OG mainly bound to Sudlow site II was further confirmed. When OG was freed from HSA and BSA, the fluorescence enhancement decreased rapidly, indicating that the formation of a stable complex was an important prerequisite for efficient fluorescence enhancement. The binding modelling was then established using the CDOCKER docking program, a molecular dynamic simulatedannealing-based algorithm implemented in DS 3.1 (Wu, Robertson, Brooks, & Vieth, 2003). OG was docked with the Sudlow site II in 2BXG. The detail docking parameters and processes used were similar to those in our previous work (Wang, Ma, He, Li, & Li, 2015). The pose with the lowest CDOCKER energy was chosen as the most suitable pose for the subsequent pose analysis. Fig. 5 shows that OG inserted well into the hydrophobic cavity of Sudlow site II. OG formed four hydrogen bonds because of its strong donor and acceptor groups. H-bonding during OG–HSA complex formation can effectively balance the adverse energetic effects of water displacement. p–cation interaction occurs between a positively charged molecule and a p system. In the OG–HSA interaction, Arg-410 acted as cation participating in the p–cation interaction. Those interactions were necessary for the formation of a stable OG–HSA complex. Dey et al. found that when the OH group was replaced by the methoxy group, the fluorescence of their probes showed very weak enhancement. Strong H-bonding was therefore necessary in optical signaling (Dey, Gaur, Giri, & Ghosh, 2016). The location of OG in HSA was relatively far from the Trp-214 residue, which is primarily attributed to the HSA emission. OG quenched HSA from a long distance. Both static quenching (ground-state
Fig. 4. The 1H (a) STD spectrum for free OG, (b) WATERGATE and (c) STD spectrum for the OG-HSA interaction and effect of (d) WF/(e)IP on the STD spectrum of the OG-HSA interaction at 298 K (pH = 7.40). The on-resonance irradiation was performed at a chemical shift of 0.7 ppm, whereas the off-resonance irradiation was conducted at 45 ppm. C(HSA) = 0.01 mM, C(OG) = 0.4 mM and C(WF/IP) = 0.8 mM. (f) Effect of PB/IP on the fluorescence of OG–HSA interaction (kex = 320 nm). C(HSA) = C(OB) = C(PB/IP) = 4 lM.
complex formation) and dynamic quenching require molecular contact between the fluorophore and quencher (Lakowicz, 2009). FRET results from long-range dipole–dipole interaction between a donor and an acceptor molecule. Thus, FRET was further confirmed to cause the interesting phenomenon in OG–HSA interaction. 3.3. Possibility for quantitative OG recognition Fluorescence-based assays offer widespread applications in the fluorescence imaging of various analytes owing to their rapid, nondestructive, selective, and sensitive measurement of emission signals (Guo, Park, Yoon, & Shin, 2014). Foods added with antioxidants include fats and oils, dairy produce, cereals, snacks, and food flavourings. The possibility for quantitative OG recognition in margarine and edible oil by using HSA was then investigated. OG has not been officially approved as a food additive in China. The acetonitrile extracts of margarine (sample A) and edible oil (sample B) used in this study did not contain OG and nearly showed no fluorescence at kex = 280, 320 nm (Fig. S5). The presence of sample A/B (4‰, v/v) did not affect the intrinsic fluorescence of HSA (4 lM). By contrast, the fluorescence enhancement of
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Fig. 5. Molecular modelling of OG docked to Sudlow site II of HSA.
Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the National Development and Reform Commission and Education of China (Grant No. 2014BW011). Thanks to School of Life Science, University of Science and Technology of China for NMR technical assistance. Appendix A. Supplementary data
Fig. 6. Relative fluorescence intensity of OG (0.14–1.69 mg/ml) in HSA (4 lM) when in the presence of 4‰ (v/v) acetonitrile extract of margarine (j, R2 = 0.983) or edible oil (d, R2 = 0.991).
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2016. 09.157. References
OG (4 lM) was inhibited at different degrees (kex = 320 nm). The inhibition ability of sample B was much stronger than that of sample A. The samples (4‰, v/v) mixed with HSA (4 lM) were spiked with different OG concentrations. A good linear correlation between the relative fluorescence enhancement of OG and its amount was observed (Fig. 6). HSA can be applied to determine OG concentration. However, foods contain a considerably low amount of OG. The use of standard addition method is more appropriate to better quantify OG. Many ligands can bind to HSA as it contains multiple hydrophobic binding sites (Vaya, LhiaubetVallet, Jimenez, & Miranda, 2014). Other non-fluorescent components existing in samples A and B more or less interfered with the fluorescence enhancement of OG. Those components may have hindered the basic complexation or the electromagnetic coupling. Hence, the standard curve for quantitative recognition of OG should be built in corresponding blank samples.
4. Conclusions In summary, the selective and sensitive fluorescent property of OG was extensively analyzed. The fluorescence of OG was obviously enhanced in the presence of HSA. After performing multiple investigations and analyses, it can be concluded that this phenomenon was a FRET phenomenon. The formation of a stable complex was an important prerequisite for efficient fluorescence enhancement. A special structure of donor, such as HSA, was also a prerequisite in fluorescence enhancement. The selective and sensitive fluorescent property of OG can be used to determine OG concentration through standard addition method, which must be performed under certain conditions.
Anees, P., Sreejith, S., & Ajayaghosh, A. (2014). Self-assembled near-infrared dye nanoparticles as a selective protein sensor by activation of a dormant fluorophore. Journal of the American Chemical Society, 136(38), 13233–13239. Aruoma, O. I., Murcia, A., Butler, J., & Halliwell, B. (1993). Evaluation of the antioxidant and prooxidant actions of gallic acid and its derivatives. Journal of the American Chemical Society, 41(11), 1880–1885. Chakraborty, J., Das, N., & Halder, U. C. (2011). Unfolding diminishes fluorescence resonance energy transfer (FRET) of lysine modified beta-lactoglobulin: Relevance towards anti-HIV binding. Journal of Photochemistry and Photobiology B: Biology, 102(1), 1–10. Dey, G., Gaur, P., Giri, R., & Ghosh, S. (2016). Optical signaling in biofluids: a nondenaturing photostable molecular probe for serum albumins. Chemical Communications, 52(9), 1887–1890. Efsa, ANS Panel (EFSA Panel on Food Additives and Nutrient Sources Added to Food) (2015). Scientific Opinion on the re-evaluation of octyl gallate (E 311) as a food additive. European Food Safety Authority Journal, 13(10), 4248. Förster, T. (1948). Zwischenmolekulare energiewanderung und fluoreszenz. Annalen der Physik Berlin, 437(1–2), 55–75. Förster, T. (1960). Transfer mechanisms of electronic excitation energy. Radiation Research Supplements, 326–339. Gelamo, E. L., Silva, C. H. T. P., Imasato, H., & Tabak, M. (2002). Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling. BBA-Protein Structure, 1594(1), 84–99. Ghosh, S., Dolai, S., Patra, T., & Dey, J. (2015). Solution behavior and interaction of pepsin with carnitine based cationic surfactant: fluorescence, circular dichroism, and calorimetric studies. The Journal of Physical Chemistry B, 119 (39), 12632–12643. Ghuman, J., Zunszain, P. A., Petitpas, I., Bhattacharya, A. A., Otagiri, M., & Curry, S. (2005). Structural basis of the drug-binding specificity of human serum albumin. Journal of Molecular Biology, 353(1), 38–52. Guo, Z. Q., Park, S., Yoon, J. Y., & Shin, I. (2014). Recent progress in the development of near-infrared fluorescent probes for bioimaging applications. Chemical Society Reviews, 43(1), 16–29. He, X. M., & Carter, D. C. (1992). Atomic structure and chemistry of human serum albumin. Nature, 358(16), 209–215. He, X. P., Zang, Y., James, T. D., Li, J., & Chen, G. R. (2015). Probing disease-related proteins with fluorogenic composite materials. Chemical Society Reviews, 44, 4239–4248. Irache, J. M., Vega, F. A., & Ezpeleta, I. (1991). Antioxidants in some pharmaceuticals, cosmetics and food from the European market. Pharmaceutica Acta Helvetiae, 67 (5–6), 152–155.
Q. Wang et al. / Food Chemistry 219 (2017) 268–273 Joint FAO/WHO Expert Committee on Food Additives (1962). Evaluation of the toxicity of a number of antimicrobials and antioxidants. World Health Organization Technical Report Series, 228, 61–65. Lakowicz, J. R. (2009). Principles of fluorescence spectroscopy (3rd ed.). Singapore: Springer. Li, X. Q., Chao, J., Sun, Y. Y., Yang, M. L., & Chu, X. G. (2009). Analysis of synthetic antioxidants and preservatives in edible vegetable oil by HPLC/TOF-MS. Food Chemistry, 113(2), 692–700. Liu, J., Chen, Y., Wang, W., Feng, J., Liang, M., Ma, S., & Chen, X. (2016). ‘‘Switch-On” fluorescent sensing of ascorbic acid in food samples based on carbon quantum dots-MnO2 probe. Journal of Agriculture and Food Chemistry, 64(1), 371–380. Majorek, K. A., Porebski, P. J., Dayal, A., Zimmerman, M. D., Jablonska, K., Stewart, A. J., ... Minor, W. (2012). Structural and immunologic characterization of bovine, horse, and rabbit serum albumins. Molecular Immunology, 52(3), 174–182. Mayer, M., & Meyer, B. (2001). Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. Journal of the American Chemical Society, 123(25), 6108–6117. Meyer, B., & Peters, T. (2003). NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angewandte Chemie International Edition, 42(8), 864–890. Mudgal, S., Keresztes, I., Feigenson, G. W., & Rizvi, S. S. (2016). Controlling the taste receptor accessible structure of rebaudioside A via binding to bovine serum albumin. Food Chemistry, 197, 84–91. Nienhaus, G. U. (2005). Protein-ligand interactions. USA: Springer. Perrin, C., & Meyer, L. (2002). Quantification of synthetic phenolic antioxidants in dry foods by reversed-phase HPLC with photodiode array detection. Food Chemistry, 77(1), 93–100. Ray, K., Badugu, R., & Lakowicz, J. R. (2015). Bloch surface wave-coupled emission from quantum dots by ensemble and single molecule spectroscopy. RSC Advance, 5(67), 54403–54411. Sen, T., Sadhu, S., & Patra, A. (2007). Surface energy transfer from rhodamine 6G to gold nanoparticles: A spectroscopic ruler. Applied Physics Letters, 91(4), 043104. Shahidi, F., Janitha, P. K., & Wanasundara, P. D. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition, 32(1), 67–103. Sudlow, G., Birkett, D. J., & Wade, D. N. (1976). Further characterization of specific drug binding sites on human serum albumin. Molecular Pharmacology, 12(6), 1052–1061.
273
Tatlidil, D., Ucuncu, M., & Akdogan, Y. (2015). Physiological concentrations of albumin favor drug binding. Physical Chemistry Chemical Physics, 17(35), 22678–22685. Vaya, I., Lhiaubet-Vallet, V., Jimenez, M. C., & Miranda, M. A. (2014). Photoactive assemblies of organic compounds and biomolecules: Drug-protein supramolecular systems. Chemical Society Reviews, 43(12), 4102–4122. Wagstaff, J. L., Taylor, S. L., & Howard, M. J. (2013). Recent developments and applications of saturation transfer difference nuclear magnetic resonance (STD NMR) spectroscopy. Molecular BioSystems, 9(4), 571–577. Wang, Q., Ma, X. L., He, J. W., Li, Y. Z., & Li, H. (2015). Insights into the fatty acid ester norethisterone enanthate binding to human albumin: fluorescence, circular dichroism, and docking investigations. RSC Advance, 5, 44696–44704. Wang, J. Y., Wu, H. L., Chen, Y., Sun, Y. M., Yu, Y. J., Zhang, X. H., & Yu, R. Q. (2012). Fast analysis of synthetic antioxidants in edible vegetable oil using trilinear component modeling of liquid chromatography–diode array detection data. Journal of Chromatography A, 1264, 63–71. Wu, G., Robertson, D. H., Brooks, C. L., & Vieth, M. (2003). Detailed analysis of grid-based molecular docking: A case study of CDOCKER—A CHARMm-based MD docking algorithm. Journal of Computational Chemistry, 24(13), 1549–1562. Wu, L., Zhao, W., Yang, R., & Yan, W. (2015). Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system. Food Chemistry, 175, 115–120. Yoshizaki, F., Oshima, T., & Imahori, K. (1971). Studies on phosphoglucomutase from an extreme thermophile, flavobacterium thermophilum HB8. Journal of Biochemistry, 69(6), 1083–1089. Yun, C. S., Javier, A., Jennings, T., Fisher, M., Hira, S., Peterson, S., ... Strouse, G. F. (2005). Nanometal surface energy transfer in optical rulers, breaking the FRET barrier. Journal of the American Chemical Society, 127(9), 3115–3119. Zhang, R., Sun, T., Liu, C. G., Song, W., Cao, Z. Z., & Liu, R. T. (2015). New insights into the toxicity of n-butanol to trypsin: Spectroscopic and molecular docking descriptions. Journal of Biochemical and Molecular Toxicology, 29(9), 418–425. Zhu, T., Du, J. J., Cao, W. B., Fan, J. L., & Peng, X. J. (2016). Microenvironment-sensitive fluorescent dyes for recognition of serum albumin in urine and imaging in living cells. Industrial and Engineering Chemistry Research, 55(3), 527–533.