- Email: [email protected]
Spectroscopic study of gamma irradiation effect on the molecular structure of bovine serum albumin
Vacuum 136 (2017) 91e96
Contents lists available at ScienceDirect
Vacuum journal homepage: www.elsevier.com/locate/vacuum
Spectroscopic study of gamma irradiation effect on the molecular structure of bovine serum albumin Hajar Zarei a, Mostean Bahreinipour a, *, Khadijeh Eskandari b, Seyed-Ali MousaviZarandi a, Susan Kaboudanian Ardestani c a b c
Department of Energy Engineering and Physics, Faculty of Physics, Amirkabir University of Technology, Tehran, Iran Nanobiotechnology Research Center, Baqiyatallah University of Medical Science, Tehran, Iran Immunology Laboratory, Institute of Biochemistry and Biophysics, University of Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 September 2016 Received in revised form 11 November 2016 Accepted 12 November 2016 Available online 24 November 2016
The effect of gamma radiation on the molecular structure, size distribution and surface charge of bovine serum albumin (BSA) was studied by spectroscopic techniques. The first structure of non-irradiated and irradiated BSA were investigated by UV-Vis spectroscopy and electrophoresis (SDSe PAGE). Additionally, the secondary and tertiary structural changes of BSA were studied by circular dichroism (CD) and fluorescence spectroscopy, respectively. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) was used for clarify of size and surface charge of BSA. The results indicate that the first structure of BSA is preserved, while the secondary and tertiary structures have changed significantly. The result of CD studies shows an 8% decrease in a-helix and an increase in other secondary structures for irradiated BSA in comparison to non-irradiated ones. Moreover, DLS and ELS displayed the decrease in the size and surface charge of irradiated BSA. The aggregation of irradiated BSA was also confirmed by fluorescence spectroscopy. The ELS results as an additional data confirm the aggregation of protein. The results demonstrate that doses close to therapeutic ones can lead to structural changes in macromolecules as well as the aggregation of polypeptide chain but without the fragmentation. © 2016 Published by Elsevier Ltd.
Keywords: Irradiation Bovine serum albumin Spectroscopy Electrophoresis Dynamic light scattering Electrophoretic light scattering
1. Introduction Proteins are important biomolecules that play different roles in selectivity and specificity of living organisms. Their conformations are prone to change under a variety of external conditions such as chemical alterations (pH, metal ions and denaturants) and as well as physical alterations (radiations and temperature), which consequently affected the protein function [1e4]. Among the different proteins in living organism, bovine serum albumin (BSA) is particularly interesting as it holds certain advantages. BSA is a single-chain transporting protein which has molecular weight around ~66 kDa [4] and the number of amino acids of BSA has frequently been cited between 582 and 607 according to literatures [5,6]. BSA has several important physiological and pharmacological functions. It transports metals, fatty acids, cholesterol, pigments, and drugs. It is a key element in the regulation of osmotic pressure
* Corresponding author. E-mail address: [email protected] (M. Bahreinipour). http://dx.doi.org/10.1016/j.vacuum.2016.11.029 0042-207X/© 2016 Published by Elsevier Ltd.
and distribution of fluid between different compartments [4]. Radiations as the physical alteration can damage cells in two ways; i) direct effect, through damaging DNA and other cellular targets, ii) indirect effect, through producing reactive oxygen species (ROS) [7]. ROS contain hydroxyl radicals (the most damaging), superoxide anion radicals, hydrogen peroxide and other oxidants which are generated by different environmental stress such as radiation. Exposure of biological organism in ionized radiation, caused ROS production in issue environment [8]. Since 70% volume of biological organism composed of water molecules that radiolysis via absorbing radiation energy and produce ROS. Generally, ROS make biological damage to vital cellular bio-molecules such as DNA, proteins and lipids [9]. Biological damages initiate by free radicals and proceeded through a variety of mechanisms. One of the damaging mechanism is cell membrane damage which is created by lipid peroxidation and protein aggregation [10]. The chemical changes that caused by irradiation to biopolymers such as proteins, leads to fragmentation, cross-linking, aggregation, and oxidation radicals generated by the radiolysis of water [11e13]. H. Schuessler and et al. [14], have reported that BSA was cleaved by the oxidative destruction of proline residues. These studies show
92
H. Zarei et al. / Vacuum 136 (2017) 91e96
that, after irradiation of proteins in solution, covalent crosslinkages are formed between free amino acids or peptides [15]. Determination and analysis of the secondary structure of proteins is investigated using FTIR [16], ATR-FTIR [17], ATR-IR [5], nuclear magnetic resonance (NMR) [18], Far UV region (190e250 nm) circular dichroism (CD) [19,20], Vacuum-Ultraviolet CD (VUVCD) [21] and Raman spectroscopy methods [22]. The near UV region (240e360 nm) CD and Fluorescence spectroscopy can provide information of tertiary structure. Changes in the local environment of tryptophan residues can be followed by changes in the emission spectra [23]. CD have limitation in ultraviolet (VUV) region between 100 and 200 nm because of the strong absorption of light by oxygen at these wavelengths. The short-wavelength limit of CD spectroscopy can be extended by the new generation of CD equipped with vacuum. Vacuum Ultraviolet CD (VUVCD) spectrophotometers using synchrotron radiation as an intense light source was developed to spread the short-wavelength limit [24e26]. The secondary structures of 15 globular proteins were investigated in the wavelength region from 160 to 260 nm under a high vacuum by VUVCD [27]. Matsuo et al. elucidated the structure of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and desaturated statues [28]. Although, the application of VUVCD recently is growing, conventional CD is still a common method. In this study, we investigated the effect of gamma radiation in the absorbed dose of 5 Gy on the BSA as the most abundant protein in blood plasma. The first, secondary and tertiary structures of BSA, as well as its size distribution and surface charge were studied. UVVis spectroscopy, Dynamic light scattering (DLS), electrophoretic light scattering (ELS), circular dichroism (CD), electrophoresis (SDSePAGE), and fluorescence spectroscopy are used to monitor structure changes of BSA. In this study, we investigated the effect of gamma radiation in the absorbed dose of 5 Gy on the BSA as the most abundant protein in blood plasma. Additionally, the secondary and tertiary structural changes of BSA were studied by circular dichroism (CD) and fluorescence spectroscopy, respectively. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) was used for clarify of size and surface charge of BSA. 2. Materials and experiments
investigation of the first protein structure. Each BSA sample was prepared at least three times and then scanned by Uv-vis, CD and fluorescence spectrophotometer. 2.2. Sample irradiation One-milligram of BSA was dissolved in 1 ml of 10 mM phosphate buffer (pH 7.0) and it was placed in glass vials and irradiated at room temperature using 60Co g-rays with dose rate of 2.68 Gy/sec, in atomic energy organization (Tehran, Iran). In this process, BSA absorbed gamma-radiation at dose of 5 Gy. 2.3. UV-Vis spectroscopy UV optical spectra of irradiated and non-irradiated BSA were recorded at room temperature in a 1-cm quartz cell by DU 800 spectrophotometer in the wavelength region 200e400 nm. The reported UV spectra were the average of three scans that the error in the reading was less than 1% (Standard error estimation). 2.4. Fluorescence spectroscopy The fluorescence emission intensity of the irradiated BSA was measured using a spectrofluorometer equipped with 1.0 cm quartz cells and a thermostat bath. The BSA solutions were excited at 280 nm and the emission spectra were recorded from 300 to 440 nm. 2.5. Circular dichroism spectroscopy (CD) The CD absorption spectra were recorded on a circular dichroism Spectrometer under nitrogen atmosphere. Quartz cells have path length of 1 and 0.1 cm for near and far region respectively. The protein concentrations were 0.2 and 0.5 mg/ml for far and near region, respectively. All the experiments were run at room temperature. The scanning speed was 200 nm/min. The CD measurements of irradiated BSA solutions were made in the range of 190e260 nm. All of the CD spectra were baseline-subtracted by using a spectrum of the solvent obtained under the same experimental conditions. Data analyzed using CDNN 2.1 (Nerve Network) software.
2.1. Reagents and apparatus 2.6. DLS and ELS measurements Bovine serum albumin (BSA) (used without further purification), tetramethylethylendiamine (TEMED), acrylamide, acrylamide-bis, tris base, coomassie brilliant blue, bromophenol blue, methanol, acetic acid, sodium chloride (NaCl), sodium hydroxide (NaOH), 2-mercaptoethanol, ammonium persulfate, isopropyl alcohol, iso-butanol, ethylene diamine tetra acetic acid (EDTA), glycerol, glycine, potassium dihydrogen phosphate (KH2PO4) and potassium hydrogen phosphate (K2HPO4), acetonitrile were purchased from Merck (Darmstadt, Germany). Sodium dodecyl sulfate and Formic acid were purchased from Sigma chemical company (USA). The solutions were prepared in deionized double distilled water (Barnstead, Nano pure infinity, USA) and all experiments were carried out at room temperature. Spectroscopic measurements were performed using UV-Vis spectrophotometer (Carry 100, Varian, Australia), spectrofluorometer (Carry edipse, varian, Australia), Circular Dichroism Spectrometer Model-215 (215, Aviv, USA). Zeta sizer Nano, ZS, (Malvern Instruments, the United Kingdom) was used for surface charge determination and measuring of the relative sizes of the BSA before and after radiation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (Sino Biological Inc, Korea) and HPLC, hypersil-bds-c18-hplc-columns-4 125 mm was applied for
DLS experiments were carried out at room temperature with a Zeta sizer Nano ZS. For each sample; the spectra were recorded with 30 scans at a time. The scattering intensity data were processed using the instrumental software to obtain the hydrodynamic diameter and the size distribution of sample. DLS data processing is dependent to fluctuation of scattered light intensity by dissolved particles at a fixed scattering angle. All experiments were performed at a q ¼ 90 and l ¼ 514 nm. ELS measurements were made on a Zeta sizer Nano ZS at room temperature. For each sample, the spectra were recorded with 100 scans at a time. 2.7. Electrophoretic analysis Protein damage was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSe PAGE). Electrophoresis of proteins was performed on 12% acrylamide resolving gel with 6% acrylamide stacking gel that was poured on top of the resolving gel. In the upper part of the stacking gel, the gel comb was placed 40 ml of each protein solution samples were mixed with sample buffer and were boiled in water for 4 min, then each of the samples in the
H. Zarei et al. / Vacuum 136 (2017) 91e96
93
Fig. 1. UVeVis absorption spectra of BSA irradiated at 5 Gy g-ray (dashed line) and not irradiated (solid line) in the PBS (10 mM, pH ¼ 7) at room temperature. RSDs based on three replicate analyses.
wells caused the gel was placed. Acrylamide gel running buffer containing 25 mM Trisbuffer, 192 mM Gly and 0.1% SDS, pH 8.3 were used. Electrophoresis was performed at 30 mA during 90 min. Gels were stained with 0.1% coomassie brilliant blue at 37 C during 2 h and destined with a solution of methanol and acetic acid during 15 h.
2.8. BSA preparation for HPLC HPLC, hypersil-bds-c18-hplc-columns-4 125 mm, d ¼ 130 Å was used. In this step, 100 mL (1 mg/ml) BSA was filtered in 900 mL formic acid (0.1%).
3. Results 3.1. UV absorption spectroscopy, SDS-PAGE and HPLC The conformation state of non-radiated and irradiated BSA was investigated by UVeVis absorption spectroscopy in the wavelength region 200e400 nm at room temperature (Fig. 1). Two peaks was revealed at around 220 nm and 276 nm for both samples (nonradiated and irradiated BSA) [29,30]. Figs. 2 and 3 display SDS-PAGE profiles and HPLC diagrams for both of non-irradiated and irradiated BSA, respectively. SDS-PAGE is a sensitive method and has been used to monitor gamma irradiation mediated protein damage. Gamma irradiation may cause breakage of the polypeptide chains through oxidative damage of amino acids in peptide chains, and protein damage can be visualized by the formation of fragmented bands and smearing of gel [13,31].
Fig. 2. Representative SDS-PAGE profile of BSA irradiated at dose 5 Gy and not irradiated. The concentration of BSA was 1 mg/ml, pH ¼ 7 at room temperature.
3.3. Fluorescence and near-UV CD spectroscopy Average fluorescence spectra (Fig. 6) of irradiated BSA (at 5 Gy) and non-irradiated BSA were obtained to evaluate conformational changes around Trp residues. The changes in the tertiary structure of the protein can be determined from the fluctuations of the fluorescence [32e34]. The fluorescence emission for BSA recorded from 300 to 440 in lex ¼ 280 nm, with maximum peak at 345 nm. Fig. 7 shows the near-UV CD spectrum of BSA with two minima around 285 nm. The near-UV CD spectra of protein are highly sensitive to interactions between nearby groups and potentially hold valuable conformational information about the protein. 3.4. ELS measurements Zeta potential (x) is the electrostatic potential, which is related to both surface charge and the local environment of the particle. Using Zeta potential analyzer, surface charge of non-radiated and
3.2. Far-UV CD spectroscopy The secondary structure of protein can be investigated by far CD spectroscopy. In this section, the molar ellipticity of non-radiated and irradiated BSA solutions was measured between 190 and 260 nm. According to Fig. 4, the far-UV CD spectra of BSA exhibited a signal characteristic of a-helix structure with two negative bands in the far-UV region at 208 and 222 nm and a positive band at 192 nm. The b-sheet structure of BSA protein also shows a negative band centered at 216 nm [30].
Fig. 3. HPLC for analyses samples BSA using UV detector. The concentration of BSA was 0.1 mg/ml, at room temperature. All solutions were prepared in the PBS (10 mM, pH ¼ 7) with a BSA concentration of 1 mg/ml.
94
H. Zarei et al. / Vacuum 136 (2017) 91e96
Fig. 4. Far-UV CD spectra of BSA not irradiated (solid line) and 1 h after irradiation at 5 Gy g-ray (dashed line). The concentration of BSA was 0.2 mg/ml in PBS (0.01 mol L-1, pH ¼ 7).
irradiated BSA at 5 Gy were recorded at room temperature. Zeta potential could automatically be calculated from electrophoretic mobility based on the Smoluchowski equation:
y ¼ ðεE=hÞ
(1)
where y is the measured electrophoretic velocity, h is viscosity, ε is the electrical permittivity of the electrolytic solution and E is the electric field [35]. 3.5. DLS measurements In order to monitor of hydrodynamic size in irradiated BSA, the DLS measurements were performed (Fig. 9). 4. Discussion 4.1. Investigation of the first protein structure by UVeVis spectroscopy, SDS-PAGE and HPLC techniques In general, UVeVis spectroscopy of protein have two absorbance regions; i) The peak around 280 nm (near-UV) is due to p/p* transferring of aromatic amino acids i.e. Trp, Tyr and Phe [4,36] and ii) The peak between 180 and 230 nm (far-UV) is attributed to peptide groups of the protein [37]. As shown in Fig. 1, in both of non-radiated and irradiated BSA samples, not significant changes was occurred in peaks of 220 nm and 276 nm. It is shows, gammaradiation in dose of 5 Gy was in effective on peptide bond in BSA. For confirmation of UVeVis result, SDS-PAGE technique was used, too. Uv-vis spectroscopy tests were done three times. The obtained spectra in both irradiated and non-irradiated BSA show a difference lower than 0.1% to each other revealing the data is reproducible. As displayed in Fig. 2, gamma-radiation in dose of 5 Gy does not significantly damage BSA natural structure. Also, this result was confirmed by HPLC technique (Fig. 3).
Table 1 Secondary structural content of BSA irradiated at 5 Gy and non-irradiated obtained from deconvolution of CD spectra in the far-UV region (190e260 nm) using the deconvolution software CDNN2.1. Solution
a-helix (%)
beta-sheet (%)
b-turn (%)
Random. Coil (%)
Nonirradiation Irradiation
56.5 48.4
8.5 10.8
13.5 14.6
19.0 23.0
Fig. 5. Far-UV CD spectra of BSA, one (solid line) and four (dashed line) hour after irradiation at 5 Gy g-ray. The concentration of BSA was 0.2 mg/ml in PBS (0.01 mol L-1, pH ¼ 7).
4.2. Investigation of the secondary structure of protein by far CD spectroscopy The reasonable explanation was that the negative peaks between 208 and 222 nm were both contributed to n/p* transfer for the peptide bond in a-helix [38]. According to Fig. 4, the far-UV CD curves of BSA with and without irradiation were similar in shape, indicating that a-helix was still the main components of BSA secondary structure. The corresponding band intensity of BSA which reflects the amount of helix in the protein decreased with the irradiation, suggesting the loss of a-helical structures and unfolding of BSA. The far CD spectra of the BSA at pH 7 were further analyzed by the algorithm CDNN2.1. The fraction contents of a-helix, b-turn, b-sheet and random forms for BSA with and without irradiation were shown in Table 1. With the BSA irradiated at 5 Gy, a decreasing of a-helices and combined with an increasing tendency of b-sheets, b-turn and random coil structure were observed. Analysis of far-UV spectra, which corresponds to the secondary structure of BSA, showed a decrease in alpha helix (8%) and an increase in beta-sheet (2.33%), beta-turn (1.07%) and random coil (3.9%) structures. These results indicate that the irradiation induced obvious changes in secondary structure of BSA. The results repeated three times of far CD spectroscopy is also reproducible and the difference is around 0.2% to each other. The influence of time on reversibility or irreversibility of secondary structure changes occurred in protein was also investigated by far-UV CD. As shown in Fig. 5, the changes of the secondary structure occurred in irradiated BSA after 4 h is irreversible. It reveals that protein is not able to deal with free radical induced damage.
4.3. Investigation of the tertiary structure of protein by fluorescence and near-UV CD spectroscopies Fig. 6 shows that, g-irradiation caused a significant decrease in the emission intensity of BSA due to the change in the local environment around tryptophan and tyrosine residues [39]. In generally, in hydrophobic environment (buried within the core of the protein), tryptophan and tyrosine have a high quantum yield and therefore high fluorescence intensity. In contrast, in a hydrophilic environment (exposed to solvent) their quantum yield decreases leading to low fluorescence intensity. Therefore, radiation caused to
H. Zarei et al. / Vacuum 136 (2017) 91e96
95
Fig. 6. Fluorescence emission spectra of BSA irradiated at an excitation wavelength of 280 nm. The concentration of BSA was 1 mg/ml, pH ¼ 7 at room temperature.
exposing of local environment around tryptophan and tyrosine residues to solvent [40]. To provide more evidence for the conformational change induced by BSA irradiated, the near-UV CD spectra were further applied too. As shown in Fig. 7, the near-UV CD spectrum of BSA showed two minima around 285 nm, characteristic of the disulfide and aromatic chromospheres, and reflected the tertiary conformation of the protein as shown in fluorescence spectroscopy [4]. The studies of tertiary structure are reliable and haven't significant difference. The influence of time on reversibility or irreversibility of changes occurred in tertiary structure of irradiated BSA was investigated by florescence. Fig. 8 shows that the modifications of tertiary structure occurred in irradiated BSA after 4 h is irreversible as mentioned about secondary structure. It reveals that environment surrounding tryptophan is not returned to its original state as a native protein.
Fig. 8. Fluorescence emission spectra of BSA, one (solid line) and 4 h (dashed line) after irradiation at 5 Gy g-ray at an excitation wavelength of 280 nm. The concentration of BSA was 1 mg/ml in PBS (0.01 mol L1, pH ¼ 7).
which indicates that the BSA solution contained more negatively charged amino acids than positively charged amino acids. Results of zeta potential measurements seem that gamma-radiation causes markedly changes in protein structure and conformation. The effective surface charge of the particles primarily determines their dispersion and aggregation [41]. Since, the surface charge of BSA was decreased, so the number of charged residues that are present at the surface of the BSA was decreased. The absolute zeta potentials of samples irradiated might have decreased because of the formation of aggregation when the samples were subjected gamma-irradiation [42]. Therefore, the irradiation caused debilitation of inter-particle electrostatic repulsions, which induce instability of protein.
4.4. Zeta potential measurements 4.5. Monitoring of hydrodynamic size by DLS The Zeta potential of non-radiated BSA was 14.7 ± 6.3 mV (n ¼ 100), that shifted to 4.17 ± 5.91 mV (n ¼ 100) in irradiated BSA. The x-potentials of all samples examined here were negative,
Fig. 7. Near-UV CD spectra of BSA, one (solid line) and four (dashed line) hour after irradiation at 5 Gy g-ray. The concentration of BSA was 0.5 mg/ml in PBS (0.01 mol L-1, pH ¼ 7).
As shown in Fig. 9, the size of non-radiated and irradiated BSA was 36.74 and 47.89 nm respectively. As shown in fluorescence spectroscopy radiation caused to exposing of local environment residues to solvent, thus water molecules can be permeate into BSA and protein hydrodynamic size was increased.
Fig. 9. The measured size of non-radiated and irradiated BSA by DLS.
96
H. Zarei et al. / Vacuum 136 (2017) 91e96
5. Conclusion The spectroscopic methods including UV-Vis, CD, fluorescence as well as electrophoresis (SDSePAGE) and HPLC technique were used to monitor structure changes of BSA. The DLS and ELS were also used to clarify of size and surface charge of BSA. The UV-Vis spectroscopy and electrophoresis (SDSe PAGE) results show that, the first structure of BSA hasn't clear change. Also, CD and fluorescence spectroscopy revealed that the changes in secondary and tertiary structures were obvious. Moreover, DLS and ELS displayed that, the size of irradiated BSA was increased and the surface charge was decreased showing the aggregation of irradiated BSA was occurred. The results demonstrate that doses close to therapeutic ones can lead to irreversible structural changes in macromolecules as well as the aggregation of polypeptide chain but without the fragmentation. Additionally, this study shows that the obtained results of studied BSA protein as a basic model can be extended to other main protein macromolecules in plasma and tissues. References [1] P.A. Fields, Review: protein function at thermal extremes: balancing stability and flexibility, Comp. Biochem. Physiology Part A Mol. Integr. Physiology 129 (2) (2001) 417e431. [2] A. Krisko, M. Radman, Protein damage and death by radiation in Escherichia coli and Deinococcus radiodurans, Proc. Natl. Acad. Sci. 107 (32) (2010) 14373e14377. [3] U. Anand, C. Jash, S. Mukherjee, Spectroscopic probing of the microenvironment in a Protein surfactant assembly, J. Phys. Chem. B 114 (48) (2010) 15839e15845. [4] M. Chen, Y. Liu, H. Cao, L. Song, Q. Zhang, The secondary and aggregation structural changes of BSA induced by trivalent chromium: a biophysical study, J. Luminescence 158 (2015) 116e124. [5] A. Bouhekka, T. Bürgi, In situ ATR-IR spectroscopy study of adsorbed protein: visible light denaturation of bovine serum albumin on TiO 2, Appl. Surf. Sci. 261 (2012) 369e374. [6] D. Silva, C.M. Cortez, J. Cunha-Bastos, S.R. Louro, Methyl parathion interaction with human and bovine serum albumin, Toxicol. Lett. 147 (1) (2004) 53e61. [7] M. Abdou, O. Abbas, Evaluation of diphenyl dimethyl bicarboxylate (DDB) as a probable hepato-protector in rats against whole body gamma irradiation, J. Biosci. Res. 6 (2009) 1e11. [8] C. Borek, Antioxidants and radiation therapy, J. Nutr. 134 (11) (2004) 3207Se3209S. [9] N. Madhu, Radioprotective effect of sulphydryl group containing triazole derivative to modulate the radiation-induced clastogenic effects, Res. Pharm. Sci. 9 (1) (2013) 23e29. [10] E.M. Kirilova, I. Kalnina, T. Zvagule, N. Gabruseva, N. Kurjane, I.I. Solomenikova, Fluorescent study of human blood plasma albumin alterations induced by ionizing radiation, J. Fluoresc. 21 (3) (2011) 923e927. [11] M.H. Gaber, Effect of g-irradiation on the molecular properties of bovine serum albumin, J. Biosci. Bioeng. 100 (2) (2005) 203e206. [12] J. Mayo, D. Tan, R. Sainz, M. Natarajan, S. Lopez-Burillo, R. Reiter, Protection against oxidative protein damage induced by metal-catalyzed reaction or alkylperoxyl radicals: comparative effects of melatonin and other antioxidants, Biochimica Biophysica Acta (BBA)-General Subj. 1620 (1) (2003) 139e150. [13] K. Mishra, H. Ojha, S. Kallepalli, A. Alok, N.K. Chaudhury, Protective effect of ferulic acid on ionizing radiation induced damage in bovine serum albumin, Int. J. Radiat. Res. 12 (2) (2014) 113e121. [14] H. Schuessler, K. Schilling, Oxygen effect in the radiolysis of proteins: Part 2 bovine serum albumin, Int. J. Radiat. Biol. 45 (3) (1984) 267e281. [15] W.M. Garrison, Reaction mechanisms in the radiolysis of peptides, polypeptides, and proteins, Chem. Rev. 87 (2) (1987) 381e398. [16] Y. Jiang, C. Li, X. Nguyen, S. Muzammil, E. Towers, J. Gabrielson, L. Narhi, Qualification of FTIR spectroscopic method for protein secondary structural analysis, J. Pharm. Sci. 100 (11) (2011) 4631e4641. [17] Y. Wang, R.I. Boysen, B.R. Wood, M. Kansiz, D. McNaughton, M.T. Hearn, Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regression, Biopolymers 89 (11) (2008) 895e905.
[18] M. Pfuhl, M. Gautel, A.S. Politou, C. Joseph, A. Pastore, Secondary structure determination by NMR spectroscopy of an immunoglobulin-like domain from the giant muscle protein titin, Journal of biomolecular NMR 6 (1) (1995) 48e58. [19] N.N. Fathima, M. Balaraman, J.R. Rao, B.U. Nair, Effect of zirconium (IV) complexes on the thermal and enzymatic stability of type I collagen, J. Inorg. Biochem. 95 (1) (2003) 47e54. [20] S.B. Corchnoy, T.E. Swartz, J.W. Lewis, I. Szundi, W.R. Briggs, R.A. Bogomolni, Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1, J. Biol. Chem. 278 (2) (2003) 724e731. [21] K. Matsuo, R. Yonehara, K. Gekko, Improved estimation of the secondary structures of proteins by vacuum-ultraviolet circular dichroism spectroscopy, J. Biochem. 138 (1) (2005) 79e88. [22] N.C. Maiti, M.M. Apetri, M.G. Zagorski, P.R. Carey, V.E. Anderson, Raman spectroscopic characterization of secondary structure in natively unfolded proteins: a-synuclein, J. Am. Chem. Soc. 126 (8) (2004) 2399e2408. [23] R.W. Henkens, B.B. Kitchell, S.C. Lottich, P.J. Stein, T.J. Williams, Detection and characterization using circular dichroism and fluorescence spectroscopy of a stable intermediate conformation formed in the denaturation of bovine carbonic anhydrase with guanidinium chloride, Biochemistry 21 (23) (1982) 5918e5923. [24] J. Sutherland, A. Emrick, L. France, D. Monteleone, J. Trunk, Circular dichroism user facility at the National Synchrotron Light Source: estimation of protein secondary structure, Biotechniques 13 (4) (1992) 588e590. [25] G.R. Jones, D.T. Clarke, Applications of extended ultra-violet circular dichroism spectroscopy in biology and medicine, Faraday Discuss. 126 (2004) 223e236. [26] B. Wallace, F. Wien, A.J. Miles, J.G. Lees, S.V. Hoffmann, P. Evans, G.J. Wistow, C. Slingsby, Biomedical applications of synchrotron radiation circular dichroism spectroscopy: identification of mutant proteins associated with disease and development of a reference database for fold motifs, Faraday Discuss. 126 (2004) 237e243. [27] K. Matsuo, R. Yonehara, K. Gekko, Secondary-structure analysis of proteins by vacuum-ultraviolet circular dichroism spectroscopy, J. Biochem. 135 (3) (2004) 405e411. [28] K. Matsuo, Y. Sakurada, R. Yonehara, M. Kataoka, K. Gekko, Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy, Biophysical J. 92 (11) (2007) 4088e4096. [29] H. Xu, N. Yao, H. Xu, T. Wang, G. Li, Z. Li, Characterization of the interaction between eupatorin and bovine serum albumin by spectroscopic and molecular modeling methods, Int. J. Mol. Sci. 14 (7) (2013) 14185e14203. [30] J. Chen, Y. Wang, Q. Zeng, X. Ding, Y. Huang, Partition of proteins with extraction in aqueous two-phase system by hydroxyl ammonium-based ionic liquid, Anal. Methods 6 (12) (2014) 4067e4076. [31] Y. Lee, K.B. Song, Effect of gamma-irradiation on the molecular properties of myoglobin, Journal of biochemistry and molecular biology 35 (6) (2002) 590e594. [32] W. Liu, Z.-Q. Zhang, C.-M. Liu, M.-Y. Xie, Z.-C. Tu, J.-H. Liu, R.-H. Liang, The effect of dynamic high-pressure microfluidization on the activity, stability and conformation of trypsin, Food Chem. 123 (3) (2010) 616e621. [33] L. Shen, C.-H. Tang, Microfluidization as a potential technique to modify surface properties of soy protein isolate, Food Res. Int. 48 (1) (2012) 108e118. [34] J.T. Vivian, P.R. Callis, Mechanisms of tryptophan fluorescence shifts in proteins, Biophysical J. 80 (5) (2001) 2093e2109. [35] K. Eskandari, H. Ghourchian, Structural changes of glucose oxidase upon interaction with gold-coated magnetic nano-particles, Int. J. Biol. Macromol. 51 (5) (2012) 998e1002. [36] Z. Chi, R. Liu, Phenotypic characterization of the binding of tetracycline to human serum albumin, Biomacromolecules 12 (1) (2010) 203e209. [37] F.X. Schmid, Biological macromolecules: UV-visible spectrophotometry, Encycl. Life Sci. (2001) 1e4. [38] D. Charbonneau, M. Beauregard, H.-A. Tajmir-Riahi, Structural analysis of human serum albumin complexes with cationic lipids, J. Phys. Chem. B 113 (6) (2009) 1777e1784. [39] M.P. Pant, J. Mariam, A. Joshi, P. Dongre, UV radiation sensitivity of bovine serum albumin bound to silver nanoparticles, J. Radiat. Res. Appl. Sci. 7 (4) (2014) 399e405. [40] S. Zolghadri, A. Saboury, E. Amin, A. Moosavi-Movahedi, A spectroscopic study on the interaction between ferric oxide nanoparticles and human hemoglobin, J. Iran. Chem. Soc. 7 (2) (2010) S145eS153. [41] X. Song, C. Zhou, F. Fu, Z. Chen, Q. Wu, Effect of high-pressure homogenization on particle size and film properties of soy protein isolate, Industrial Crops Prod. 43 (2013) 538e544. [42] L. Jiang, J. Wang, Y. Li, Z. Wang, J. Liang, R. Wang, Y. Chen, W. Ma, B. Qi, M. Zhang, Effects of ultrasound on the structure and physical properties of black bean protein isolates, Food Res. Int. 62 (2014) 595e601.
Contents lists available at ScienceDirect
Vacuum journal homepage: www.elsevier.com/locate/vacuum
Spectroscopic study of gamma irradiation effect on the molecular structure of bovine serum albumin Hajar Zarei a, Mostean Bahreinipour a, *, Khadijeh Eskandari b, Seyed-Ali MousaviZarandi a, Susan Kaboudanian Ardestani c a b c
Department of Energy Engineering and Physics, Faculty of Physics, Amirkabir University of Technology, Tehran, Iran Nanobiotechnology Research Center, Baqiyatallah University of Medical Science, Tehran, Iran Immunology Laboratory, Institute of Biochemistry and Biophysics, University of Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 September 2016 Received in revised form 11 November 2016 Accepted 12 November 2016 Available online 24 November 2016
The effect of gamma radiation on the molecular structure, size distribution and surface charge of bovine serum albumin (BSA) was studied by spectroscopic techniques. The first structure of non-irradiated and irradiated BSA were investigated by UV-Vis spectroscopy and electrophoresis (SDSe PAGE). Additionally, the secondary and tertiary structural changes of BSA were studied by circular dichroism (CD) and fluorescence spectroscopy, respectively. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) was used for clarify of size and surface charge of BSA. The results indicate that the first structure of BSA is preserved, while the secondary and tertiary structures have changed significantly. The result of CD studies shows an 8% decrease in a-helix and an increase in other secondary structures for irradiated BSA in comparison to non-irradiated ones. Moreover, DLS and ELS displayed the decrease in the size and surface charge of irradiated BSA. The aggregation of irradiated BSA was also confirmed by fluorescence spectroscopy. The ELS results as an additional data confirm the aggregation of protein. The results demonstrate that doses close to therapeutic ones can lead to structural changes in macromolecules as well as the aggregation of polypeptide chain but without the fragmentation. © 2016 Published by Elsevier Ltd.
Keywords: Irradiation Bovine serum albumin Spectroscopy Electrophoresis Dynamic light scattering Electrophoretic light scattering
1. Introduction Proteins are important biomolecules that play different roles in selectivity and specificity of living organisms. Their conformations are prone to change under a variety of external conditions such as chemical alterations (pH, metal ions and denaturants) and as well as physical alterations (radiations and temperature), which consequently affected the protein function [1e4]. Among the different proteins in living organism, bovine serum albumin (BSA) is particularly interesting as it holds certain advantages. BSA is a single-chain transporting protein which has molecular weight around ~66 kDa [4] and the number of amino acids of BSA has frequently been cited between 582 and 607 according to literatures [5,6]. BSA has several important physiological and pharmacological functions. It transports metals, fatty acids, cholesterol, pigments, and drugs. It is a key element in the regulation of osmotic pressure
* Corresponding author. E-mail address: [email protected] (M. Bahreinipour). http://dx.doi.org/10.1016/j.vacuum.2016.11.029 0042-207X/© 2016 Published by Elsevier Ltd.
and distribution of fluid between different compartments [4]. Radiations as the physical alteration can damage cells in two ways; i) direct effect, through damaging DNA and other cellular targets, ii) indirect effect, through producing reactive oxygen species (ROS) [7]. ROS contain hydroxyl radicals (the most damaging), superoxide anion radicals, hydrogen peroxide and other oxidants which are generated by different environmental stress such as radiation. Exposure of biological organism in ionized radiation, caused ROS production in issue environment [8]. Since 70% volume of biological organism composed of water molecules that radiolysis via absorbing radiation energy and produce ROS. Generally, ROS make biological damage to vital cellular bio-molecules such as DNA, proteins and lipids [9]. Biological damages initiate by free radicals and proceeded through a variety of mechanisms. One of the damaging mechanism is cell membrane damage which is created by lipid peroxidation and protein aggregation [10]. The chemical changes that caused by irradiation to biopolymers such as proteins, leads to fragmentation, cross-linking, aggregation, and oxidation radicals generated by the radiolysis of water [11e13]. H. Schuessler and et al. [14], have reported that BSA was cleaved by the oxidative destruction of proline residues. These studies show
92
H. Zarei et al. / Vacuum 136 (2017) 91e96
that, after irradiation of proteins in solution, covalent crosslinkages are formed between free amino acids or peptides [15]. Determination and analysis of the secondary structure of proteins is investigated using FTIR [16], ATR-FTIR [17], ATR-IR [5], nuclear magnetic resonance (NMR) [18], Far UV region (190e250 nm) circular dichroism (CD) [19,20], Vacuum-Ultraviolet CD (VUVCD) [21] and Raman spectroscopy methods [22]. The near UV region (240e360 nm) CD and Fluorescence spectroscopy can provide information of tertiary structure. Changes in the local environment of tryptophan residues can be followed by changes in the emission spectra [23]. CD have limitation in ultraviolet (VUV) region between 100 and 200 nm because of the strong absorption of light by oxygen at these wavelengths. The short-wavelength limit of CD spectroscopy can be extended by the new generation of CD equipped with vacuum. Vacuum Ultraviolet CD (VUVCD) spectrophotometers using synchrotron radiation as an intense light source was developed to spread the short-wavelength limit [24e26]. The secondary structures of 15 globular proteins were investigated in the wavelength region from 160 to 260 nm under a high vacuum by VUVCD [27]. Matsuo et al. elucidated the structure of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and desaturated statues [28]. Although, the application of VUVCD recently is growing, conventional CD is still a common method. In this study, we investigated the effect of gamma radiation in the absorbed dose of 5 Gy on the BSA as the most abundant protein in blood plasma. The first, secondary and tertiary structures of BSA, as well as its size distribution and surface charge were studied. UVVis spectroscopy, Dynamic light scattering (DLS), electrophoretic light scattering (ELS), circular dichroism (CD), electrophoresis (SDSePAGE), and fluorescence spectroscopy are used to monitor structure changes of BSA. In this study, we investigated the effect of gamma radiation in the absorbed dose of 5 Gy on the BSA as the most abundant protein in blood plasma. Additionally, the secondary and tertiary structural changes of BSA were studied by circular dichroism (CD) and fluorescence spectroscopy, respectively. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) was used for clarify of size and surface charge of BSA. 2. Materials and experiments
investigation of the first protein structure. Each BSA sample was prepared at least three times and then scanned by Uv-vis, CD and fluorescence spectrophotometer. 2.2. Sample irradiation One-milligram of BSA was dissolved in 1 ml of 10 mM phosphate buffer (pH 7.0) and it was placed in glass vials and irradiated at room temperature using 60Co g-rays with dose rate of 2.68 Gy/sec, in atomic energy organization (Tehran, Iran). In this process, BSA absorbed gamma-radiation at dose of 5 Gy. 2.3. UV-Vis spectroscopy UV optical spectra of irradiated and non-irradiated BSA were recorded at room temperature in a 1-cm quartz cell by DU 800 spectrophotometer in the wavelength region 200e400 nm. The reported UV spectra were the average of three scans that the error in the reading was less than 1% (Standard error estimation). 2.4. Fluorescence spectroscopy The fluorescence emission intensity of the irradiated BSA was measured using a spectrofluorometer equipped with 1.0 cm quartz cells and a thermostat bath. The BSA solutions were excited at 280 nm and the emission spectra were recorded from 300 to 440 nm. 2.5. Circular dichroism spectroscopy (CD) The CD absorption spectra were recorded on a circular dichroism Spectrometer under nitrogen atmosphere. Quartz cells have path length of 1 and 0.1 cm for near and far region respectively. The protein concentrations were 0.2 and 0.5 mg/ml for far and near region, respectively. All the experiments were run at room temperature. The scanning speed was 200 nm/min. The CD measurements of irradiated BSA solutions were made in the range of 190e260 nm. All of the CD spectra were baseline-subtracted by using a spectrum of the solvent obtained under the same experimental conditions. Data analyzed using CDNN 2.1 (Nerve Network) software.
2.1. Reagents and apparatus 2.6. DLS and ELS measurements Bovine serum albumin (BSA) (used without further purification), tetramethylethylendiamine (TEMED), acrylamide, acrylamide-bis, tris base, coomassie brilliant blue, bromophenol blue, methanol, acetic acid, sodium chloride (NaCl), sodium hydroxide (NaOH), 2-mercaptoethanol, ammonium persulfate, isopropyl alcohol, iso-butanol, ethylene diamine tetra acetic acid (EDTA), glycerol, glycine, potassium dihydrogen phosphate (KH2PO4) and potassium hydrogen phosphate (K2HPO4), acetonitrile were purchased from Merck (Darmstadt, Germany). Sodium dodecyl sulfate and Formic acid were purchased from Sigma chemical company (USA). The solutions were prepared in deionized double distilled water (Barnstead, Nano pure infinity, USA) and all experiments were carried out at room temperature. Spectroscopic measurements were performed using UV-Vis spectrophotometer (Carry 100, Varian, Australia), spectrofluorometer (Carry edipse, varian, Australia), Circular Dichroism Spectrometer Model-215 (215, Aviv, USA). Zeta sizer Nano, ZS, (Malvern Instruments, the United Kingdom) was used for surface charge determination and measuring of the relative sizes of the BSA before and after radiation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (Sino Biological Inc, Korea) and HPLC, hypersil-bds-c18-hplc-columns-4 125 mm was applied for
DLS experiments were carried out at room temperature with a Zeta sizer Nano ZS. For each sample; the spectra were recorded with 30 scans at a time. The scattering intensity data were processed using the instrumental software to obtain the hydrodynamic diameter and the size distribution of sample. DLS data processing is dependent to fluctuation of scattered light intensity by dissolved particles at a fixed scattering angle. All experiments were performed at a q ¼ 90 and l ¼ 514 nm. ELS measurements were made on a Zeta sizer Nano ZS at room temperature. For each sample, the spectra were recorded with 100 scans at a time. 2.7. Electrophoretic analysis Protein damage was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSe PAGE). Electrophoresis of proteins was performed on 12% acrylamide resolving gel with 6% acrylamide stacking gel that was poured on top of the resolving gel. In the upper part of the stacking gel, the gel comb was placed 40 ml of each protein solution samples were mixed with sample buffer and were boiled in water for 4 min, then each of the samples in the
H. Zarei et al. / Vacuum 136 (2017) 91e96
93
Fig. 1. UVeVis absorption spectra of BSA irradiated at 5 Gy g-ray (dashed line) and not irradiated (solid line) in the PBS (10 mM, pH ¼ 7) at room temperature. RSDs based on three replicate analyses.
wells caused the gel was placed. Acrylamide gel running buffer containing 25 mM Trisbuffer, 192 mM Gly and 0.1% SDS, pH 8.3 were used. Electrophoresis was performed at 30 mA during 90 min. Gels were stained with 0.1% coomassie brilliant blue at 37 C during 2 h and destined with a solution of methanol and acetic acid during 15 h.
2.8. BSA preparation for HPLC HPLC, hypersil-bds-c18-hplc-columns-4 125 mm, d ¼ 130 Å was used. In this step, 100 mL (1 mg/ml) BSA was filtered in 900 mL formic acid (0.1%).
3. Results 3.1. UV absorption spectroscopy, SDS-PAGE and HPLC The conformation state of non-radiated and irradiated BSA was investigated by UVeVis absorption spectroscopy in the wavelength region 200e400 nm at room temperature (Fig. 1). Two peaks was revealed at around 220 nm and 276 nm for both samples (nonradiated and irradiated BSA) [29,30]. Figs. 2 and 3 display SDS-PAGE profiles and HPLC diagrams for both of non-irradiated and irradiated BSA, respectively. SDS-PAGE is a sensitive method and has been used to monitor gamma irradiation mediated protein damage. Gamma irradiation may cause breakage of the polypeptide chains through oxidative damage of amino acids in peptide chains, and protein damage can be visualized by the formation of fragmented bands and smearing of gel [13,31].
Fig. 2. Representative SDS-PAGE profile of BSA irradiated at dose 5 Gy and not irradiated. The concentration of BSA was 1 mg/ml, pH ¼ 7 at room temperature.
3.3. Fluorescence and near-UV CD spectroscopy Average fluorescence spectra (Fig. 6) of irradiated BSA (at 5 Gy) and non-irradiated BSA were obtained to evaluate conformational changes around Trp residues. The changes in the tertiary structure of the protein can be determined from the fluctuations of the fluorescence [32e34]. The fluorescence emission for BSA recorded from 300 to 440 in lex ¼ 280 nm, with maximum peak at 345 nm. Fig. 7 shows the near-UV CD spectrum of BSA with two minima around 285 nm. The near-UV CD spectra of protein are highly sensitive to interactions between nearby groups and potentially hold valuable conformational information about the protein. 3.4. ELS measurements Zeta potential (x) is the electrostatic potential, which is related to both surface charge and the local environment of the particle. Using Zeta potential analyzer, surface charge of non-radiated and
3.2. Far-UV CD spectroscopy The secondary structure of protein can be investigated by far CD spectroscopy. In this section, the molar ellipticity of non-radiated and irradiated BSA solutions was measured between 190 and 260 nm. According to Fig. 4, the far-UV CD spectra of BSA exhibited a signal characteristic of a-helix structure with two negative bands in the far-UV region at 208 and 222 nm and a positive band at 192 nm. The b-sheet structure of BSA protein also shows a negative band centered at 216 nm [30].
Fig. 3. HPLC for analyses samples BSA using UV detector. The concentration of BSA was 0.1 mg/ml, at room temperature. All solutions were prepared in the PBS (10 mM, pH ¼ 7) with a BSA concentration of 1 mg/ml.
94
H. Zarei et al. / Vacuum 136 (2017) 91e96
Fig. 4. Far-UV CD spectra of BSA not irradiated (solid line) and 1 h after irradiation at 5 Gy g-ray (dashed line). The concentration of BSA was 0.2 mg/ml in PBS (0.01 mol L-1, pH ¼ 7).
irradiated BSA at 5 Gy were recorded at room temperature. Zeta potential could automatically be calculated from electrophoretic mobility based on the Smoluchowski equation:
y ¼ ðεE=hÞ
(1)
where y is the measured electrophoretic velocity, h is viscosity, ε is the electrical permittivity of the electrolytic solution and E is the electric field [35]. 3.5. DLS measurements In order to monitor of hydrodynamic size in irradiated BSA, the DLS measurements were performed (Fig. 9). 4. Discussion 4.1. Investigation of the first protein structure by UVeVis spectroscopy, SDS-PAGE and HPLC techniques In general, UVeVis spectroscopy of protein have two absorbance regions; i) The peak around 280 nm (near-UV) is due to p/p* transferring of aromatic amino acids i.e. Trp, Tyr and Phe [4,36] and ii) The peak between 180 and 230 nm (far-UV) is attributed to peptide groups of the protein [37]. As shown in Fig. 1, in both of non-radiated and irradiated BSA samples, not significant changes was occurred in peaks of 220 nm and 276 nm. It is shows, gammaradiation in dose of 5 Gy was in effective on peptide bond in BSA. For confirmation of UVeVis result, SDS-PAGE technique was used, too. Uv-vis spectroscopy tests were done three times. The obtained spectra in both irradiated and non-irradiated BSA show a difference lower than 0.1% to each other revealing the data is reproducible. As displayed in Fig. 2, gamma-radiation in dose of 5 Gy does not significantly damage BSA natural structure. Also, this result was confirmed by HPLC technique (Fig. 3).
Table 1 Secondary structural content of BSA irradiated at 5 Gy and non-irradiated obtained from deconvolution of CD spectra in the far-UV region (190e260 nm) using the deconvolution software CDNN2.1. Solution
a-helix (%)
beta-sheet (%)
b-turn (%)
Random. Coil (%)
Nonirradiation Irradiation
56.5 48.4
8.5 10.8
13.5 14.6
19.0 23.0
Fig. 5. Far-UV CD spectra of BSA, one (solid line) and four (dashed line) hour after irradiation at 5 Gy g-ray. The concentration of BSA was 0.2 mg/ml in PBS (0.01 mol L-1, pH ¼ 7).
4.2. Investigation of the secondary structure of protein by far CD spectroscopy The reasonable explanation was that the negative peaks between 208 and 222 nm were both contributed to n/p* transfer for the peptide bond in a-helix [38]. According to Fig. 4, the far-UV CD curves of BSA with and without irradiation were similar in shape, indicating that a-helix was still the main components of BSA secondary structure. The corresponding band intensity of BSA which reflects the amount of helix in the protein decreased with the irradiation, suggesting the loss of a-helical structures and unfolding of BSA. The far CD spectra of the BSA at pH 7 were further analyzed by the algorithm CDNN2.1. The fraction contents of a-helix, b-turn, b-sheet and random forms for BSA with and without irradiation were shown in Table 1. With the BSA irradiated at 5 Gy, a decreasing of a-helices and combined with an increasing tendency of b-sheets, b-turn and random coil structure were observed. Analysis of far-UV spectra, which corresponds to the secondary structure of BSA, showed a decrease in alpha helix (8%) and an increase in beta-sheet (2.33%), beta-turn (1.07%) and random coil (3.9%) structures. These results indicate that the irradiation induced obvious changes in secondary structure of BSA. The results repeated three times of far CD spectroscopy is also reproducible and the difference is around 0.2% to each other. The influence of time on reversibility or irreversibility of secondary structure changes occurred in protein was also investigated by far-UV CD. As shown in Fig. 5, the changes of the secondary structure occurred in irradiated BSA after 4 h is irreversible. It reveals that protein is not able to deal with free radical induced damage.
4.3. Investigation of the tertiary structure of protein by fluorescence and near-UV CD spectroscopies Fig. 6 shows that, g-irradiation caused a significant decrease in the emission intensity of BSA due to the change in the local environment around tryptophan and tyrosine residues [39]. In generally, in hydrophobic environment (buried within the core of the protein), tryptophan and tyrosine have a high quantum yield and therefore high fluorescence intensity. In contrast, in a hydrophilic environment (exposed to solvent) their quantum yield decreases leading to low fluorescence intensity. Therefore, radiation caused to
H. Zarei et al. / Vacuum 136 (2017) 91e96
95
Fig. 6. Fluorescence emission spectra of BSA irradiated at an excitation wavelength of 280 nm. The concentration of BSA was 1 mg/ml, pH ¼ 7 at room temperature.
exposing of local environment around tryptophan and tyrosine residues to solvent [40]. To provide more evidence for the conformational change induced by BSA irradiated, the near-UV CD spectra were further applied too. As shown in Fig. 7, the near-UV CD spectrum of BSA showed two minima around 285 nm, characteristic of the disulfide and aromatic chromospheres, and reflected the tertiary conformation of the protein as shown in fluorescence spectroscopy [4]. The studies of tertiary structure are reliable and haven't significant difference. The influence of time on reversibility or irreversibility of changes occurred in tertiary structure of irradiated BSA was investigated by florescence. Fig. 8 shows that the modifications of tertiary structure occurred in irradiated BSA after 4 h is irreversible as mentioned about secondary structure. It reveals that environment surrounding tryptophan is not returned to its original state as a native protein.
Fig. 8. Fluorescence emission spectra of BSA, one (solid line) and 4 h (dashed line) after irradiation at 5 Gy g-ray at an excitation wavelength of 280 nm. The concentration of BSA was 1 mg/ml in PBS (0.01 mol L1, pH ¼ 7).
which indicates that the BSA solution contained more negatively charged amino acids than positively charged amino acids. Results of zeta potential measurements seem that gamma-radiation causes markedly changes in protein structure and conformation. The effective surface charge of the particles primarily determines their dispersion and aggregation [41]. Since, the surface charge of BSA was decreased, so the number of charged residues that are present at the surface of the BSA was decreased. The absolute zeta potentials of samples irradiated might have decreased because of the formation of aggregation when the samples were subjected gamma-irradiation [42]. Therefore, the irradiation caused debilitation of inter-particle electrostatic repulsions, which induce instability of protein.
4.4. Zeta potential measurements 4.5. Monitoring of hydrodynamic size by DLS The Zeta potential of non-radiated BSA was 14.7 ± 6.3 mV (n ¼ 100), that shifted to 4.17 ± 5.91 mV (n ¼ 100) in irradiated BSA. The x-potentials of all samples examined here were negative,
Fig. 7. Near-UV CD spectra of BSA, one (solid line) and four (dashed line) hour after irradiation at 5 Gy g-ray. The concentration of BSA was 0.5 mg/ml in PBS (0.01 mol L-1, pH ¼ 7).
As shown in Fig. 9, the size of non-radiated and irradiated BSA was 36.74 and 47.89 nm respectively. As shown in fluorescence spectroscopy radiation caused to exposing of local environment residues to solvent, thus water molecules can be permeate into BSA and protein hydrodynamic size was increased.
Fig. 9. The measured size of non-radiated and irradiated BSA by DLS.
96
H. Zarei et al. / Vacuum 136 (2017) 91e96
5. Conclusion The spectroscopic methods including UV-Vis, CD, fluorescence as well as electrophoresis (SDSePAGE) and HPLC technique were used to monitor structure changes of BSA. The DLS and ELS were also used to clarify of size and surface charge of BSA. The UV-Vis spectroscopy and electrophoresis (SDSe PAGE) results show that, the first structure of BSA hasn't clear change. Also, CD and fluorescence spectroscopy revealed that the changes in secondary and tertiary structures were obvious. Moreover, DLS and ELS displayed that, the size of irradiated BSA was increased and the surface charge was decreased showing the aggregation of irradiated BSA was occurred. The results demonstrate that doses close to therapeutic ones can lead to irreversible structural changes in macromolecules as well as the aggregation of polypeptide chain but without the fragmentation. Additionally, this study shows that the obtained results of studied BSA protein as a basic model can be extended to other main protein macromolecules in plasma and tissues. References [1] P.A. Fields, Review: protein function at thermal extremes: balancing stability and flexibility, Comp. Biochem. Physiology Part A Mol. Integr. Physiology 129 (2) (2001) 417e431. [2] A. Krisko, M. Radman, Protein damage and death by radiation in Escherichia coli and Deinococcus radiodurans, Proc. Natl. Acad. Sci. 107 (32) (2010) 14373e14377. [3] U. Anand, C. Jash, S. Mukherjee, Spectroscopic probing of the microenvironment in a Protein surfactant assembly, J. Phys. Chem. B 114 (48) (2010) 15839e15845. [4] M. Chen, Y. Liu, H. Cao, L. Song, Q. Zhang, The secondary and aggregation structural changes of BSA induced by trivalent chromium: a biophysical study, J. Luminescence 158 (2015) 116e124. [5] A. Bouhekka, T. Bürgi, In situ ATR-IR spectroscopy study of adsorbed protein: visible light denaturation of bovine serum albumin on TiO 2, Appl. Surf. Sci. 261 (2012) 369e374. [6] D. Silva, C.M. Cortez, J. Cunha-Bastos, S.R. Louro, Methyl parathion interaction with human and bovine serum albumin, Toxicol. Lett. 147 (1) (2004) 53e61. [7] M. Abdou, O. Abbas, Evaluation of diphenyl dimethyl bicarboxylate (DDB) as a probable hepato-protector in rats against whole body gamma irradiation, J. Biosci. Res. 6 (2009) 1e11. [8] C. Borek, Antioxidants and radiation therapy, J. Nutr. 134 (11) (2004) 3207Se3209S. [9] N. Madhu, Radioprotective effect of sulphydryl group containing triazole derivative to modulate the radiation-induced clastogenic effects, Res. Pharm. Sci. 9 (1) (2013) 23e29. [10] E.M. Kirilova, I. Kalnina, T. Zvagule, N. Gabruseva, N. Kurjane, I.I. Solomenikova, Fluorescent study of human blood plasma albumin alterations induced by ionizing radiation, J. Fluoresc. 21 (3) (2011) 923e927. [11] M.H. Gaber, Effect of g-irradiation on the molecular properties of bovine serum albumin, J. Biosci. Bioeng. 100 (2) (2005) 203e206. [12] J. Mayo, D. Tan, R. Sainz, M. Natarajan, S. Lopez-Burillo, R. Reiter, Protection against oxidative protein damage induced by metal-catalyzed reaction or alkylperoxyl radicals: comparative effects of melatonin and other antioxidants, Biochimica Biophysica Acta (BBA)-General Subj. 1620 (1) (2003) 139e150. [13] K. Mishra, H. Ojha, S. Kallepalli, A. Alok, N.K. Chaudhury, Protective effect of ferulic acid on ionizing radiation induced damage in bovine serum albumin, Int. J. Radiat. Res. 12 (2) (2014) 113e121. [14] H. Schuessler, K. Schilling, Oxygen effect in the radiolysis of proteins: Part 2 bovine serum albumin, Int. J. Radiat. Biol. 45 (3) (1984) 267e281. [15] W.M. Garrison, Reaction mechanisms in the radiolysis of peptides, polypeptides, and proteins, Chem. Rev. 87 (2) (1987) 381e398. [16] Y. Jiang, C. Li, X. Nguyen, S. Muzammil, E. Towers, J. Gabrielson, L. Narhi, Qualification of FTIR spectroscopic method for protein secondary structural analysis, J. Pharm. Sci. 100 (11) (2011) 4631e4641. [17] Y. Wang, R.I. Boysen, B.R. Wood, M. Kansiz, D. McNaughton, M.T. Hearn, Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regression, Biopolymers 89 (11) (2008) 895e905.
[18] M. Pfuhl, M. Gautel, A.S. Politou, C. Joseph, A. Pastore, Secondary structure determination by NMR spectroscopy of an immunoglobulin-like domain from the giant muscle protein titin, Journal of biomolecular NMR 6 (1) (1995) 48e58. [19] N.N. Fathima, M. Balaraman, J.R. Rao, B.U. Nair, Effect of zirconium (IV) complexes on the thermal and enzymatic stability of type I collagen, J. Inorg. Biochem. 95 (1) (2003) 47e54. [20] S.B. Corchnoy, T.E. Swartz, J.W. Lewis, I. Szundi, W.R. Briggs, R.A. Bogomolni, Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1, J. Biol. Chem. 278 (2) (2003) 724e731. [21] K. Matsuo, R. Yonehara, K. Gekko, Improved estimation of the secondary structures of proteins by vacuum-ultraviolet circular dichroism spectroscopy, J. Biochem. 138 (1) (2005) 79e88. [22] N.C. Maiti, M.M. Apetri, M.G. Zagorski, P.R. Carey, V.E. Anderson, Raman spectroscopic characterization of secondary structure in natively unfolded proteins: a-synuclein, J. Am. Chem. Soc. 126 (8) (2004) 2399e2408. [23] R.W. Henkens, B.B. Kitchell, S.C. Lottich, P.J. Stein, T.J. Williams, Detection and characterization using circular dichroism and fluorescence spectroscopy of a stable intermediate conformation formed in the denaturation of bovine carbonic anhydrase with guanidinium chloride, Biochemistry 21 (23) (1982) 5918e5923. [24] J. Sutherland, A. Emrick, L. France, D. Monteleone, J. Trunk, Circular dichroism user facility at the National Synchrotron Light Source: estimation of protein secondary structure, Biotechniques 13 (4) (1992) 588e590. [25] G.R. Jones, D.T. Clarke, Applications of extended ultra-violet circular dichroism spectroscopy in biology and medicine, Faraday Discuss. 126 (2004) 223e236. [26] B. Wallace, F. Wien, A.J. Miles, J.G. Lees, S.V. Hoffmann, P. Evans, G.J. Wistow, C. Slingsby, Biomedical applications of synchrotron radiation circular dichroism spectroscopy: identification of mutant proteins associated with disease and development of a reference database for fold motifs, Faraday Discuss. 126 (2004) 237e243. [27] K. Matsuo, R. Yonehara, K. Gekko, Secondary-structure analysis of proteins by vacuum-ultraviolet circular dichroism spectroscopy, J. Biochem. 135 (3) (2004) 405e411. [28] K. Matsuo, Y. Sakurada, R. Yonehara, M. Kataoka, K. Gekko, Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy, Biophysical J. 92 (11) (2007) 4088e4096. [29] H. Xu, N. Yao, H. Xu, T. Wang, G. Li, Z. Li, Characterization of the interaction between eupatorin and bovine serum albumin by spectroscopic and molecular modeling methods, Int. J. Mol. Sci. 14 (7) (2013) 14185e14203. [30] J. Chen, Y. Wang, Q. Zeng, X. Ding, Y. Huang, Partition of proteins with extraction in aqueous two-phase system by hydroxyl ammonium-based ionic liquid, Anal. Methods 6 (12) (2014) 4067e4076. [31] Y. Lee, K.B. Song, Effect of gamma-irradiation on the molecular properties of myoglobin, Journal of biochemistry and molecular biology 35 (6) (2002) 590e594. [32] W. Liu, Z.-Q. Zhang, C.-M. Liu, M.-Y. Xie, Z.-C. Tu, J.-H. Liu, R.-H. Liang, The effect of dynamic high-pressure microfluidization on the activity, stability and conformation of trypsin, Food Chem. 123 (3) (2010) 616e621. [33] L. Shen, C.-H. Tang, Microfluidization as a potential technique to modify surface properties of soy protein isolate, Food Res. Int. 48 (1) (2012) 108e118. [34] J.T. Vivian, P.R. Callis, Mechanisms of tryptophan fluorescence shifts in proteins, Biophysical J. 80 (5) (2001) 2093e2109. [35] K. Eskandari, H. Ghourchian, Structural changes of glucose oxidase upon interaction with gold-coated magnetic nano-particles, Int. J. Biol. Macromol. 51 (5) (2012) 998e1002. [36] Z. Chi, R. Liu, Phenotypic characterization of the binding of tetracycline to human serum albumin, Biomacromolecules 12 (1) (2010) 203e209. [37] F.X. Schmid, Biological macromolecules: UV-visible spectrophotometry, Encycl. Life Sci. (2001) 1e4. [38] D. Charbonneau, M. Beauregard, H.-A. Tajmir-Riahi, Structural analysis of human serum albumin complexes with cationic lipids, J. Phys. Chem. B 113 (6) (2009) 1777e1784. [39] M.P. Pant, J. Mariam, A. Joshi, P. Dongre, UV radiation sensitivity of bovine serum albumin bound to silver nanoparticles, J. Radiat. Res. Appl. Sci. 7 (4) (2014) 399e405. [40] S. Zolghadri, A. Saboury, E. Amin, A. Moosavi-Movahedi, A spectroscopic study on the interaction between ferric oxide nanoparticles and human hemoglobin, J. Iran. Chem. Soc. 7 (2) (2010) S145eS153. [41] X. Song, C. Zhou, F. Fu, Z. Chen, Q. Wu, Effect of high-pressure homogenization on particle size and film properties of soy protein isolate, Industrial Crops Prod. 43 (2013) 538e544. [42] L. Jiang, J. Wang, Y. Li, Z. Wang, J. Liang, R. Wang, Y. Chen, W. Ma, B. Qi, M. Zhang, Effects of ultrasound on the structure and physical properties of black bean protein isolates, Food Res. Int. 62 (2014) 595e601.