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Protection by ethyl pyruvate against gamma radiation induced damage in bovine serum albumin
Journal Pre-proofs Protection by Ethyl Pyruvate against Gamma Radiation Induced Damage in Bovine Serum Albumin Deepti Sharma, Anju Singh, Shrikant Kukreti, Mallika Pathak, Lajpreet Kaur, Vinod Kaushik, Himanshu Ojha PII: DOI: Reference:
S0141-8130(19)33506-8 https://doi.org/10.1016/j.ijbiomac.2019.10.110 BIOMAC 13611
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
12 May 2019 11 October 2019 11 October 2019
Please cite this article as: D. Sharma, A. Singh, S. Kukreti, M. Pathak, L. Kaur, V. Kaushik, H. Ojha, Protection by Ethyl Pyruvate against Gamma Radiation Induced Damage in Bovine Serum Albumin, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.110
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© 2019 Published by Elsevier B.V.
Protection by Ethyl Pyruvate against Gamma Radiation Induced Damage in Bovine Serum Albumin Deepti Sharmaa, Anju Singhb, Shrikant Kukretib, Mallika Pathakc, Lajpreet Kaura, Vinod Kaushika, Himanshu Ojhaa* a
Protection and Decontamination research group, Division of CBRN Defence, Institute of Nuclear Medicine and
Allied Sciences, Delhi-110054 b
c
Nucleic Acid Research Lab, Department of Chemistry, University of Delhi, Delhi-110007
Department of Chemistry, Miranda House, University of Delhi, Delhi-110007
*Corresponding author Dr Himanshu Ojha Scientist E Protection and Decontamination research group Division of CBRN Defence Institute of Nuclear Medicine and Allied Sciences Timarpur, Delhi 110054 Tel +91-11-23905186 Fax+91-11-23919509 Email: [email protected]
Abstract Environmental factors like ionizing radiation induced generation of reactive oxygen species (ROS) cause macromolecular damage under physiological conditions. Proteins are the potential targets of ROS induced oxidative damage because of their abundance and their critical functions in the biological systems. The present study investigates the protective potential of ethyl pyruvate (EP) against ionizing radiation induced oxidative damage of bovine serum albumin (BSA) using spectroscopic, biochemical and SDS-PAGE techniques. Spectroscopic data shows that EP prevents the build up of protein damage markers like bityrosine formation and oxidation of tryptophan. Protein melting studies shows that the melting temperature (Tm) of the irradiated protein does not change significantly in the presence of EP. Biochemical assays indicate that ionizing radiation causes the generation of carbonyls and malondialdehyde and the loss of thiol content in proteins that is prevented by EP. The SDS-PAGE profile of gamma irradiated BSA shows the radioprotective effect of EP. These results indicate the radiation induced oxidative and molecular changes in the protein and that the EP protected the BSA from these modifications. Therefore, these results imply that EP has a good antiradical property and hence it can be proposed as a good radioprotective agent. Keywords: BSA, fluorescence, bityrosine, SDS-PAGE, Carbonylation, Malondialdehyde, BSA melting
1. Introduction Exposure to ionizing radiations is one of the various environmental stresses that have detrimental effects on living organisms. Such exposure can damage macromolecules such as proteins, DNA, lipids. Proteins are the potential targets of ionizing radiation induced oxidative damage because of their abundance in cellular environment of living systems [1-3]. The extreme protein damage may leads to their accumulation and dysfunctioning which results in initiation of various diseases like Alzheimer, Parkinson, prion disease, amyloidosis, sickle cell anaemia etc [4-8]. In direct protein damage, the breakage of covalent bonds of polypeptide chains is irreversible in nature that leads to protein fragmentations into various low molecular weight proteins and peptides [9, 10]. The indirect damage to proteins through ionizing radiations occurs via the reaction of water radiolysis products with proteins. Ionizing radiations generate reactive oxygen species (ROS) in the intracellular and intercellular water through radiolysis and proteins are the main targets for these oxidative species [11]. This oxidative damage modifies the primary structure of protein leading to alterations in the secondary and tertiary structures of protein [12, 13]. Protein damage can occur due to their reaction with ROS causing their aggregation and fragmentation which leads to alteration in their structure and function [2, 10]. Various studies have proposed the use of natural antioxidants as therapeutic or prophylactic agents against protein damage occur in various pathological conditions such as vitamin D has the both prophylactic and therapeutic role in various types of cancers, vitamin E, phycocyanin, berberine, etc., can be used as a medicine in renal
diseases like
nephrolithiasis [14-19]. Ethyl pyruvate (EP) is a synthetic stable, lipophilic aliphatic ester of pyruvic acid (Fig. 1) [20, 21]. EP scavenges reactive oxygen species (ROS) effectively and render anti-inflammatory effects thereby reducing the systemic inflammation in lipopolysaccharide (LPS)-mediated
multi-organ damage by inhibiting the expression of cytokines, tumor necrosis factor-alpha (TNF-α) and high mobility group box protein 1 (HMG-B1) and inhibiting methoxyglycol (MGO)-induced cell apoptosis [20-26]. EP reduces significantly the inflammatory response of lung epithelial cells by decreasing the expression of IL-8, improving the permeability in ileal mucosa in severe hemorrhagic shock models and decrease the systemic inflammatory response resulted from shock and ischemia [27-30]. Pathak et al [31] showed that EP can bind effectively to bovine serum albumin (BSA) that indicated expected favourable biodistribution of EP. Besides, literature survey indicated that EP can significantly display prophylactic radioprotective agent and mitigating agent in the both in vitro and in vivo models [20]. However, in the literature there is not a single published study which indicated the mechanistic aspect of radioprotective potential of EP for ionizing radiation induced protein damage. There are several studies which investigated glycation mediated, irradiation and chemically induced protein damage [32, 33, 9, 10] which have selected BSA as model protein owe to its availability and well defined structure characterization. Therefore, the present study is an attempt to investigate the radioprotective effects of EP against gamma-irradiation induced damage using BSA as protein model by employing spectroscopic (UV-Vis absorption, fluorescence, circular dichroism, differential scanning calorimetry) and SDSPAGE techniques. 2. Materials and Methods 2.1. Materials Bovine serum albumin (BSA, fraction V, approximately 99%; protease free and essentially -Globulin free) was purchased from Sigma Aldrich chemical company (USA). Ethyl pyruvate (>98% purity) was purchased from Acros Organics. 2, 4-dinitro phenyl-hydrazine (DNPH), trichloroacetic acid (TCA) and Guanidine hydrochloride (GuHCl), 2-deoxy D-
ribose, thiobarbituric acid (TBA), methanol (>99.9%), acrylamide, ammonium persulphate, glacial acetic acid, Coomassie brilliant blue R 250, bromophenol blue, sodium dodecyl sulphate and tetramethylethylenediamine (TEMED) were purchased from Sigma Aldrich chemical company (USA). Sodium dihydrogen phosphate dihydrate and anhydrous disodium hydrogen phosphate were purchased from E- Merck Germany. Sodium chloride (NaCl), absolute ethanol, ethyl acetate, 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB),mercaptoethanol, tris base, tris hydrochloride, glycerol, glycine were purchased from Himedia (Mumbai, India).The hydrochloric acid was purchased from Thermo Fischer scientific (India). The water used for preparation of solutions was (18 Mega ohm MQ grade) derived from Millipore water system (Millipore Corp USA, model Elix 3). 2.2. Sample Preparation The stock and working solutions of BSA and EP were prepared in phosphate buffered saline (PBS), pH 7.4 as per the method mentioned by Pathak et al [31] in their earlier published work. The working concentration of protein sample was 114 μM for measurements of UV-Vis absorption, fluorescence emission and biochemical assays such as carbonyl formation. The working concentration of BSA was kept 75 μM for biochemical assay used to determine thiol content of protein in native and irradiated BSA. SDS-PAGE profile and CD spectroscopy studies were performed using working concentrations of BSA of 4.5 and 1 μM, respectively. 2.3. Gamma Irradiation procedure The BSA solutions were prepared in borosilicate glass vials and irradiated at two doses, 500 Gy and 1000 Gy (dose rate, 1.1 k Gy/h) using 60Co gamma ray irradiator, Model GC 5000, BRIT, Mumbai, India,). Protein samples containing 15 µM, 150 µM and 1500 µM EP was irradiated at both 500 Gy and 1000 Gy.
2.4. SDS-PAGE All the protein samples were run and analyzed using SDS-PAGE as suggested by Laemmli with some modifications [34]. 10 μl of each of the protein samples were added to 10 μl of 6.25 mM Tris HCL buffer (pH 6.8) containing 25% glycerol, 2% w/v SDS, 0.01% w/v bromophenol blue. These samples were heated for 5 mins at 95 °C and cooled in air. 20 μl of each of protein samples were loaded in the respective wells on an SDS-PAGE gel containing separating gel with 10% polyacrylamide and stacking gel with 4% polyacrylamide. The samples were electrophoresed at a constant voltage for 90 minutes (stacking at 45 V and resolving at 70 V). The gel was stained with 0.25% w/v Coomassie Brilliant Blue R 250 in water/methanol/acetic acid (9:9:2 by volume) at 25 °C with gentle shaking and destained with the destaining solution consisting of water/methanol/acetic acid (9:9:2 by volume). Gel image was captured using Gel DOC system (Biorad, USA). 2.5. UV-Vis Absorption measurement The UV-Vis absorbance measurement of protein samples (native, irradiated, pretreated with EP and irradiated) exposed to different doses that is 500 Gy and 1000 Gy was performed using UV-Vis spectrophotometer (Cary 100 BIO, Varian, Australia) equipped with a peltier thermostatic cell holder. The absorption of protein samples was measured using PBS as a reference. 3 ml of the each protein samples were kept in the 3.5 ml capacity quartz cuvette of 10 mm path length. All the samples were scanned from 200 nm - 400 nm wavelength range at 25 °C. 2.6. Fluorescence measurement Tryptophan oxidation and bityrosine formation in the protein samples were measured using spectrofluorophotometer (FS920, Edinburg instruments, Edinburg, UK). The excitation source was xenon lamp (450 W) and sample chamber was equipped with Peltier accessory. Scanning parameters for all measurements were optimized with slit width 4 nm for
excitation and emission, dwell time 0.2 s and wavelength step 0.5 nm. The measurements of tryptophan loss and bityrosine production in BSA samples were measured by keeping the protein sample (75 μM) in a 3.5 ml capacity quartz cuvette with a path length of 10 mm. The excitation wavelength was kept at 295 nm to excite selectively the tryptophan residues and emission was measured from 310 nm – 400 nm. Dityrosine content was measured in the protein samples at excitation wavelength of 325 nm and emission wavelength of 390 nm – 500 nm [35]. All the fluorescence measurements were performed at 25 °C. 2.7. Circular dichroism measurement The circular dichroism measurement was performed on spectropolarimeter (J18, Jasco, Japan) equipped with peltier accessory. The measurements were performed by the method given by Pathak et al [31]. The scan of each protein sample was taken in the wavelength range of 200 nm - 250 nm. CD melting experiment was performed on JASCO 815 spectropolarimeter interfaced with an IBM PC compatible computer, calibrated with D-Camphor Sulphonic acid. Samples were prepared by heating at 95 °C followed by gradual cooling to 25 °C. Melting data was collected at the ramp rate of 1 °C /min between the temperature ranges of 40 °C – 95 °C. Band width and data pitch were fixed to 1 nm and 0.2°C, respectively. CD melting data of native, irradiated and pretreated with EP and irradiated BSA samples to different doses of gamma radiation that is, 500 Gy and 1000 Gy, was recorded at the wavelength of 222 nm. CD measurements of all the samples were measured at 25 °C. 2.8. Protein carbonylation and DTNB assay Protein carbonyl formation of protein samples were evaluated using the methods suggested by Hawkins et al [36] with some modifications. The protein whether in the native or in irradiated without and with the treatment of EP (15 μΜ, 150 μΜ and 1500 μΜ) were incubated with equal volume of 10 mM DNPH (prepared in 2N HCl) in dark at 25 °C for 1
hour. The samples were precipitated by adding ice cold 50% TCA solution and centrifuged at 5000 rpm for 10 minutes. The pellets were washed 3 times with ethanol/ethyl acetate (1/1; v/v) solution to remove any unbound DNPH. The resulting pellet was resuspended in 1 ml 6M GuHCL. The protein samples were quantified for protein carbonylation formation at 371 nm against GuHCL as reference. The calculations were carried out according to the method given by Mishra et al [1]. In the DTNB assay, the protein samples were analyzed for the loss of thiol group in the irradiated BSA samples in the presence and absence of different concentrations of EP (15 μΜ, 150 μΜ and 1500 μΜ) by the method suggested by Hawkins et al [36] with some modifications. 1.5 ml of protein samples were mixed with 200 μl of 2 mM DTNB prepared in PBS (pH 7.4). The samples were kept for incubation period in dark for 30 minutes at 25 °C. The samples were analyzed for thiol content at 412 nm with PBS as reference using the molar extinction co-efficient of 13600 M-1 cm-1 [1]. 2.9. Hydroxyl radical scavenging assay Hydroxyl radical scavenging potential of EP was examined using 2-deoxy-D-ribose degradation assay and measured using a UV Vis spectrophotometer. 2-deoxy-D-ribose samples were prepared in 10 mM PBS, mixed with different concentrations of EP (15 μΜ, 150 μΜ and 1500 μΜ) and irradiated at radiation doses of 500 Gy and 1000 Gy. Each sample was mixed with 1 ml each of 10% TCA and 1% TBA (prepared in 0.05N NaOH) and heated at 95 oC for 30 minutes. Samples were cooled and absorbance was taken at 532 nm. 2.10. Statistical analysis The results were expressed as mean with standard error of mean (mean± SEM). The results were examined statistically by one way ANOVA with Tukey’s post hoc test analysis as
statistically significant at 95% confidence level (p<0.05) with the help of Graph Pad Prism version-5.0. 3. Results and discussion 3.1. SDS-PAGE Analysis It can be observed that the major band of native BSA was observed at 65 kDa in SDSPAGE of protein using protein markers of molecular weight ranging from 192-15 kDa (Fig. 2). The intensity of the major band of BSA was prominent in the native BSA sample, but at 500 Gy the band intensity is decreased which ultimately reduces significantly to a very low level at 1000 Gy with the lightening and light smearing of bands. Earlier, Mishra et al [1], Lee et al [9], Moon and Song [13] have shown that γ-irradiation of protein at lower doses of up to 1000 Gy causes the breakage of polypeptide chain that results in decrease of major protein band intensity with same amount of protein loaded. The various studies have shown that the loss of intensity of the major band and smearing of protein may be caused due to the degradation of polypeptide chains of protein at low radiation doses. Therefore, in the present work it is inferred that radiation exposure of 500 Gy and 1000 Gy caused significant damage in BSA. However, BSA samples pre-treated with different concentrations of EP (D1: 15 µM, D2: 150 µM, D3: 1500 µM) showed no appreciable change in the band intensity upon irradiation at 500 Gy and 1000 Gy (Fig. 2). It was observed that intensity of the major bands (65kDa) was retained as close to the intensity of native BSA in the presence of three different concentrations of EP. The observations of SDS-PAGE profile of BSA were found to be in accordance with earlier reported studies. Mishra et al [1] has showed that ferulic acid prevents the γ-irradiation induced damage of BSA by scavenging the free radicals. Similarly, Moon and Song [13] have also confirmed the protective effect of ascorbic acid against γ-radiation induced albumin damage by scavenging the reactive oxygen species. Therefore, SDS-PAGE profile of native, irradiated and pretreated with EP BSA irradiated at
different doses, 500 Gy and 1000 Gy confirmed the radioprotective effect of EP against gamma irradiation induced protein damage. 3.2. UV-Vis Spectroscopy UV-Vis spectroscopy is the most widely used biophysical technique for measuring changes in the absorbance of a molecule on binding and for assessing the damage [37]. In this study, this technique is used to determine the extent of damage produced by gamma radiation in DNA and the effect of EP in preventing that damage. In Fig. 3 (a and b), the UV- spectra of BSA shows an increase in the absorption intensity of BSA at high dose of gamma radiation, i.e., 1000 Gy but no change in the absorption intensity at low gamma radiation dose (500 Gy). There was a pioneering study which has recorded changes in the spectroscopic signals of BSA when irradiated to different doses of ionizing radiation. In the same work, the author has confirmed that there were a change in UV absorbance of BSA due to the breakage of covalent bonds of protein and disordering the protein’s structure [12]. Similar observations were recorded in the present study when BSA samples were irradiated to 500 Gy and 1000 Gy. While, the absorbance of BSA samples were retained when it was treated with different concentrations of EP prior γirradiation at 500 Gy and 1000 Gy. It was occurred because the ordered structure of BSA protein was retained in the presence of EP as it binds to BSA significantly [31]. 3.3. Fluorescence measurements 3.3.1 Fluorescence bityrosine formation When the BSA protein was irradiated with gamma radiation both at 500 Gy (Fig. 4a) and 1000 Gy (Fig. 4b), it leads to increase in the fluorescence emission intensity of protein at 425 nm. The increase in fluorescence intensity around 425 nm, in case of ionizing irradiation induced structural modification of serum protein, was due to the formation of tyrosine dimers formed by reaction of tyrosine residues of protein with water radiolysis
products, mainly ˙OH [35]. This ˙OH abstracts the hydrogen from tyrosine residues forming tyrosyl radicals which in turn reacts with other tyrosyl radicals and forms bityrosine and cross-links the protein that resulted in increase in fluorescence intensity of protein [3, 38, 39]. However, when BSA was irradiated in the presence of different concentrations of EP, the fluorescence intensity at 425 nm decreased steadily with the increase in EP concentration. The observation suggested the inhibition of bityrosine formation in the presence of EP. 3.3.2 Fluorescence tryptophan degradation The fluorescence intensity at emission wavelength ranging 339 nm - 350 nm is due to the emission from tryptophan residues when excited selectively at 295 nm [37, 40]. Therefore, fluorescence band around 339 nm is characteristic marker for tryptophan residues in BSA. The decrease in fluorescence intensity of BSA around 339 nm indicated that irradiation has damaged the tryptophan residues due to the oxidation by the ROS formed by the radiolysis of water [35]. The fluorescence emission of BSA around 339 nm decreases significantly as protein samples were irradiated with radiation doses of 500 Gy (Fig. 5a) and 1000 Gy (Fig. 5b). Fluorescence quenching of protein increases proportionally with gamma radiation doses [12, 13]. Similar to the observation in case of bityrosine formation, EP reduces the impact of gamma irradiation by retaining the tryptophan emission in protein samples when administered with EP. Besides, EP shows its protective effect in proportion to its concentration. 3.4. CD measurement CD is an extensively sensitive technique to investigate the conformational changes in protein. Different structural element of protein such as α- helix, β-sheet, random coils and turns all have characteristic CD signatures. The proteins containing α- helical content exhibit two negative CD peaks in UV region centered on 208 nm and 222 nm while having β-content shows negative band at 218 nm [41]. The negative peak centered at 208 nm arises
due to the exciton splitting of the peptide * transition whereas the peak at 220 nm originates due to n* transition [42, 43]. A CD study is performed in the wavelength range from 200 nm to 250 nm in absence as well as in presence of radiation and EP. Fig. 6 demonstrates the typical Far-UV CD spectra of BSA in the absence and presence of various gyration of radiation and EP. BSA displayed a typical CD signature with negative ellipticity centered at 208 nm and 222 nm having considerable amount of alpha helical content. In presence of EP the helicity of protein gets increased with little shifts from 208 nm peak to the 211 nm. This shows that EP interacts with BSA and stabilizes the protein by binding to it. When BSA protein alone is irradiated with gamma radiation in 500 Gy as well as 1000 Gy the helicity of protein diminishes to a greater extent. The protein structure collapses at 1000 Gy dose. A considerable reduction in magnitude of negative ellipticity of the CD peaks is observed at both wavelengths. It is clearly observed from CD spectra that peak at 208 nm and 222 nm both decrease sharply but the decrease in ellipticity was observed largely at 208 nm peak. However, after addition of EP in BSA prior gamma irradiation with 500 Gy gamma radiation, the helicity of protein is retained to a larger extent while in 1000 Gy irradiated BSA with prior addition of EP, retention of protein helicity is less. It is clearly seen from CD spectra that in presence of EP the protein retains its structure and EP protects the secondary structure of protein. Radiation alters the protein conformation and disrupts the intramolecular bonding between amino acids in protein. It is also observed from the CD studies that EP can help in retaining the conformation and protecting the secondary structure of protein from radiation damage. Further the stability was investigated by CD melting studies on BSA protein in presence of radiation and EP. Thermal stability of BSA protein is investigated by CD melting studies. CD melting experiment was performed in the range of 200 nm to 250 nm by heating at the rate of 0.5 ᵒC
in the temperature range of 40 ᵒC -95 ᵒC. CD melting spectra is displayed in Fig. 7 in the absence and presence of gamma radiation and EP. CD melting spectra clearly demonstrates that BSA protein alone show melting temperature (Tm) at approximately 55 ᵒC whereas on addition of EP, the Tm is observed somewhere around 75 ᵒC. So, it is clearly observed that EP predominantly stabilizes the protein secondary structure. Further BSA was irradiated at different doses of gamma radiation i.e. with 500 Gy and 1000 Gy. Thermal stability is again observed and it is seen from spectra that in the presence of radiation, protein structure collapses and all the factors like intramolecular forces and hydrogen bonding are disrupted due to interaction with gamma radiation. Damage is observed to a greater extent in the presence of 1000 Gy dose of radiation. It was reported in literature that unfolding of protein proceeds in various steps including native to extended and followed by unfolding. In these steps protein retain its natural folded state to some extent while in unfolding state protein starts to melt [44-46]. Further on the addition of EP in irradiated BSA, stability of protein structure is observed and it was shown by CD melting spectra that EP provides the retention of protein structure and the protein regains its conformation again. This study is in well agreement with the CD studies where the protein secondary structure is well retained in presence of EP at different doses of gamma radiation. These studies can help in understanding the interaction of various drugs with BSA protein and doses of radiation which can cause less alteration to the protein conformation. These types of studies play a pivotal role in biomedical field. 3.5. Protein carbonylation and DTNB Assay Carbonylation is the stable and irreversible transformation that damages the protein and hence its function [4, 47]. Ionizing radiation enhances the protein oxidation and causes the formation of carbonyl groups in the proteins [1]. Protein carbonyls are generated by
multiple radicals directly and by singlet oxygen species as a result of secondary reactions [36]. In Fig. 8, it is shown that the carbonyl content in BSA in the presence of two doses of gamma radiation, 500 Gy and 1000 Gy, has increased from 14.23 μM to 71.7 μM and 82.67 μM, respectively. In the presence of different concentrations of EP [15 µM (D1), 150 µM (D2) and 1500 µM (D3)], the formation of carbonyl content in BSA reduces at both the radiation doses. Mishra et al [1] has also showed in their study that the ferulic acid prevented the formation of carbonyls in the BSA after gamma irradiation. This study is in agreement with the earlier performed study and hence suggests the protective effect of EP on gamma radiation induced damage in BSA. Most of the proteins including BSA contain thiol groups as cysteine residues and oxidation of these thiol groups is an indicator of protein modification. The modification of these cysteine residues can be persuaded by many oxidants like peroxides, peroxynitrile, singlet oxygen and many radicals that can form multiple products such as cystine, mixed disulfides, etc. [36]. The loss of thiol groups from the proteins is related to loss of their activity [1]. When DTNB reacts with reduced thiols, it results in formation of 5-thio-2-nitrobenzoic acid (TNB). Gamma irradiation of BSA at 500 Gy caused loss of thiol content from 62.2 μM to 40 μM. Irradiation to 1000 Gy caused no further change in the thiol content suggesting that protein damage in terms of thiol content reached saturation at 500 Gy. But in the presence of increasing doses of EP, the loss of thiol content reduced proportionally to the presence of EP in the protein sample (Fig. 9). Hence, the assay confirmed the protective effect of EP against radiation induced thiol oxidation in protein. 3.6. Hydroxyl radical scavenging assay
Malondialdehyde (MDA) is a TBA-reactive matter that is produced by free-radical mediated destruction of carbohydrate moieties, e.g., 2-D-deoxyribose sugar, especially by hydroxyl radicals [48]. The ionizing radiations cause the hydrolysis of water that leads to the generation of hydroxyl radicals leading to DNA damage [3]. For determining the hydroxyl radical scavenging ability of EP, 2-D-deoxyribose damage was produced by hydroxyl radicals generated by ionizing radiations and examined at different concentrations of EP (125 µM to 4000 µM). A radiation dose of 500 Gy and 1000 Gy produced around 2.95
µM
and
3.82
µM
of
malondialdehyde
(MDA)
(Fig.
10).
The results suggest that EP decreased the formation of MDA in a concentration-dependent manner in samples irradiated with 500 Gy and 1000 Gy. EP at 4000 µM decreased the hydroxyl radical mediated formation of MDA by 80% and 68% at 500 Gy and 1000 Gy, respectively. 4. Conclusion The anti-radical property of EP was assessed against gamma radiation induced BSA damage through
SDS-PAGE,
spectroscopic
techniques
including
UV-Vis
spectroscopy,
fluorescence spectroscopy and CD spectroscopy and biochemical analysis such as, carbonyls formation, thiols oxidation and malondialdehyde formation in proteins. All these above studies show that when BSA is irradiated with gamma radiation in the presence of EP, the damage in BSA protein is prevented due to its radical scavenging property. Hence, EP has a therapeutic effect in protecting protein against gamma radiation and can be proposed as a better radioprotective agent. With further analysis involving in vivo and in vitro safety studies, an effective formulation either oral or topical can be prepared. These formulations can be used as prophylactic and therapeutic agents for the treatment of injuries involving radiation exposure.
Conflict of Interest There is no conflict of interest among the authors. Acknowledgements The authors are sincerely thankful to Director INMAS, DRDO for supporting and making available the facilities to carry out the research work. Authors are sincerely thankful to Head of department, Department of Chemistry, University of Delhi and Principal, Miranda House, for providing spectroscopic techniques to carry out the research. One of the authors Ms Deepti Sharma thanks UGC for providing senior research fellowship. Ms Lajpreet Kaur is thankful to CSIR for providing junior research fellowship. Figure legends Fig. 1 Molecular structure of ethyl pyruvate Fig. 2 SDS-PAGE profile of native and gamma-irradiated BSA at two doses: 500 Gy and 1000 Gy in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM]. SDS-PAGE is showing smear and almost a significant loss of BSA when irradiated at 1000 Gy, retaining the protein in the presence of EP. Similarly, at 500 Gy, the damage was low as smeared product was less but gets retained in the presence of EP. Fig. 3 a) UV-Vis spectra of irradiated BSA at (a)500 Gy and (b) 1000 Gy in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM]. The spectra indicated the negligible change in absorbance of irradiated BSA in the presence of higher concentrations of EP. Fig. 4 Fluorescence spectra of native and irradiated BSA at (a) 500 Gy; (b) 1000 Gy in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM] preventing bityrosine formation within BSA protein.
Fig. 5 Fluorescence spectra of native and irradiated BSA (a) at 500 Gy; (b) at 1000 Gy in the absence and presence of different concentrations of EP [D1: 15 µM EP; D2: 150 µM EP and D3: 1500 µM EP] preventing tryptophan degradation within BSA protein. The fluorescence intensity of tryptophan in irradiated BSA sample was found negligibly changed with the addition of EP. Fig. 6 CD spectra of native and irradiated BSA. BSA was irradiated with gamma radiation at 500 Gy and 1000 Gy in the absence and presence of EP (D3: 1500 µM) showing that EP helps in retention of protein conformation in the irradiated BSA. Fig. 7 CD melting spectra of native nd gamma irradiated BSA (500 Gy and 1000 Gy) in the presence of highest concentration of EP (D3: 1500 µM). In the presence of EP, the irradiated BSA protein retained its original Tm. Fig. 8 The graph showing the formation of carbonyls in BSA when irradiated with gamma radiation (500 Gy and 1000 Gy) in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM] (p<0.05). In the presence of EP, the concentration of carbonyls is decreasing significantly with the increasing concentration of EP. Fig. 9 The graph showing the content of thiols in BSA when irradiated with gamma radiation (500 Gy and 1000 Gy) in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM] (p<0.05). The thiol content of irradiated BSA is increasing significantly with the increasing concentration of EP. Fig. 10 The graph showing the malondialdehyde content in BSA when irradiated with gamma radiation (500 Gy and 1000 Gy) in the absence and presence of different concentrations of EP [E1: 125 µM; E2: 250 µM; E3: 500 µM; E4: 1000 µM; E5: 2000 µM; E6: 4000 µM]
(p<0.05). The malondialdehyde which is a marker of oxidative damage decreasing significantly with the increasing concentration of EP.
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Protection by Ethyl Pyruvate against Gamma Radiation Induced Damage in Bovine Serum Albumin Deepti Sharmaa, Anju Singhb, Shrikant Kukretib, Mallika Pathakc, Lajpreet Kaura, Vinod Kaushika, Himanshu Ojhaa* a
Protection and Decontamination research group, Division of CBRN Defence, Institute of Nuclear Medicine and Allied Sciences, Delhi-110054
b
c
Nucleic Acid Research Lab, Department of Chemistry, University of Delhi, Delhi-110007
Department of Chemistry, Miranda House, University of Delhi, Delhi-110007
*Corresponding author Dr Himanshu Ojha Scientist E Protection and Decontamination research group Division of CBRN Defence Institute of Nuclear Medicine and Allied Sciences Timarpur, Delhi 110054 Tel +91-11-23905186 Fax+91-11-23919509 Email: [email protected]
O O
O Fig. 1
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200
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θ deg cm2 mol-1
-20 -30
-40
BSA Control BSA +D3
-50
Irrad BSA 500
-60
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-70
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-80 -90
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-10 40
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Highlights
Radioprotective effect of ethyl pyruvate (EP) on bovine serum albumin was studied.
EP prevented bityrosine formation & tryptophan degradation.
SDS-PAGE showed low BSA protein damage in presence of EP.
Biochemical assays proved protective role of EP on protein (BSA) damage.
S0141-8130(19)33506-8 https://doi.org/10.1016/j.ijbiomac.2019.10.110 BIOMAC 13611
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
12 May 2019 11 October 2019 11 October 2019
Please cite this article as: D. Sharma, A. Singh, S. Kukreti, M. Pathak, L. Kaur, V. Kaushik, H. Ojha, Protection by Ethyl Pyruvate against Gamma Radiation Induced Damage in Bovine Serum Albumin, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.110
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Protection by Ethyl Pyruvate against Gamma Radiation Induced Damage in Bovine Serum Albumin Deepti Sharmaa, Anju Singhb, Shrikant Kukretib, Mallika Pathakc, Lajpreet Kaura, Vinod Kaushika, Himanshu Ojhaa* a
Protection and Decontamination research group, Division of CBRN Defence, Institute of Nuclear Medicine and
Allied Sciences, Delhi-110054 b
c
Nucleic Acid Research Lab, Department of Chemistry, University of Delhi, Delhi-110007
Department of Chemistry, Miranda House, University of Delhi, Delhi-110007
*Corresponding author Dr Himanshu Ojha Scientist E Protection and Decontamination research group Division of CBRN Defence Institute of Nuclear Medicine and Allied Sciences Timarpur, Delhi 110054 Tel +91-11-23905186 Fax+91-11-23919509 Email: [email protected]
Abstract Environmental factors like ionizing radiation induced generation of reactive oxygen species (ROS) cause macromolecular damage under physiological conditions. Proteins are the potential targets of ROS induced oxidative damage because of their abundance and their critical functions in the biological systems. The present study investigates the protective potential of ethyl pyruvate (EP) against ionizing radiation induced oxidative damage of bovine serum albumin (BSA) using spectroscopic, biochemical and SDS-PAGE techniques. Spectroscopic data shows that EP prevents the build up of protein damage markers like bityrosine formation and oxidation of tryptophan. Protein melting studies shows that the melting temperature (Tm) of the irradiated protein does not change significantly in the presence of EP. Biochemical assays indicate that ionizing radiation causes the generation of carbonyls and malondialdehyde and the loss of thiol content in proteins that is prevented by EP. The SDS-PAGE profile of gamma irradiated BSA shows the radioprotective effect of EP. These results indicate the radiation induced oxidative and molecular changes in the protein and that the EP protected the BSA from these modifications. Therefore, these results imply that EP has a good antiradical property and hence it can be proposed as a good radioprotective agent. Keywords: BSA, fluorescence, bityrosine, SDS-PAGE, Carbonylation, Malondialdehyde, BSA melting
1. Introduction Exposure to ionizing radiations is one of the various environmental stresses that have detrimental effects on living organisms. Such exposure can damage macromolecules such as proteins, DNA, lipids. Proteins are the potential targets of ionizing radiation induced oxidative damage because of their abundance in cellular environment of living systems [1-3]. The extreme protein damage may leads to their accumulation and dysfunctioning which results in initiation of various diseases like Alzheimer, Parkinson, prion disease, amyloidosis, sickle cell anaemia etc [4-8]. In direct protein damage, the breakage of covalent bonds of polypeptide chains is irreversible in nature that leads to protein fragmentations into various low molecular weight proteins and peptides [9, 10]. The indirect damage to proteins through ionizing radiations occurs via the reaction of water radiolysis products with proteins. Ionizing radiations generate reactive oxygen species (ROS) in the intracellular and intercellular water through radiolysis and proteins are the main targets for these oxidative species [11]. This oxidative damage modifies the primary structure of protein leading to alterations in the secondary and tertiary structures of protein [12, 13]. Protein damage can occur due to their reaction with ROS causing their aggregation and fragmentation which leads to alteration in their structure and function [2, 10]. Various studies have proposed the use of natural antioxidants as therapeutic or prophylactic agents against protein damage occur in various pathological conditions such as vitamin D has the both prophylactic and therapeutic role in various types of cancers, vitamin E, phycocyanin, berberine, etc., can be used as a medicine in renal
diseases like
nephrolithiasis [14-19]. Ethyl pyruvate (EP) is a synthetic stable, lipophilic aliphatic ester of pyruvic acid (Fig. 1) [20, 21]. EP scavenges reactive oxygen species (ROS) effectively and render anti-inflammatory effects thereby reducing the systemic inflammation in lipopolysaccharide (LPS)-mediated
multi-organ damage by inhibiting the expression of cytokines, tumor necrosis factor-alpha (TNF-α) and high mobility group box protein 1 (HMG-B1) and inhibiting methoxyglycol (MGO)-induced cell apoptosis [20-26]. EP reduces significantly the inflammatory response of lung epithelial cells by decreasing the expression of IL-8, improving the permeability in ileal mucosa in severe hemorrhagic shock models and decrease the systemic inflammatory response resulted from shock and ischemia [27-30]. Pathak et al [31] showed that EP can bind effectively to bovine serum albumin (BSA) that indicated expected favourable biodistribution of EP. Besides, literature survey indicated that EP can significantly display prophylactic radioprotective agent and mitigating agent in the both in vitro and in vivo models [20]. However, in the literature there is not a single published study which indicated the mechanistic aspect of radioprotective potential of EP for ionizing radiation induced protein damage. There are several studies which investigated glycation mediated, irradiation and chemically induced protein damage [32, 33, 9, 10] which have selected BSA as model protein owe to its availability and well defined structure characterization. Therefore, the present study is an attempt to investigate the radioprotective effects of EP against gamma-irradiation induced damage using BSA as protein model by employing spectroscopic (UV-Vis absorption, fluorescence, circular dichroism, differential scanning calorimetry) and SDSPAGE techniques. 2. Materials and Methods 2.1. Materials Bovine serum albumin (BSA, fraction V, approximately 99%; protease free and essentially -Globulin free) was purchased from Sigma Aldrich chemical company (USA). Ethyl pyruvate (>98% purity) was purchased from Acros Organics. 2, 4-dinitro phenyl-hydrazine (DNPH), trichloroacetic acid (TCA) and Guanidine hydrochloride (GuHCl), 2-deoxy D-
ribose, thiobarbituric acid (TBA), methanol (>99.9%), acrylamide, ammonium persulphate, glacial acetic acid, Coomassie brilliant blue R 250, bromophenol blue, sodium dodecyl sulphate and tetramethylethylenediamine (TEMED) were purchased from Sigma Aldrich chemical company (USA). Sodium dihydrogen phosphate dihydrate and anhydrous disodium hydrogen phosphate were purchased from E- Merck Germany. Sodium chloride (NaCl), absolute ethanol, ethyl acetate, 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB),mercaptoethanol, tris base, tris hydrochloride, glycerol, glycine were purchased from Himedia (Mumbai, India).The hydrochloric acid was purchased from Thermo Fischer scientific (India). The water used for preparation of solutions was (18 Mega ohm MQ grade) derived from Millipore water system (Millipore Corp USA, model Elix 3). 2.2. Sample Preparation The stock and working solutions of BSA and EP were prepared in phosphate buffered saline (PBS), pH 7.4 as per the method mentioned by Pathak et al [31] in their earlier published work. The working concentration of protein sample was 114 μM for measurements of UV-Vis absorption, fluorescence emission and biochemical assays such as carbonyl formation. The working concentration of BSA was kept 75 μM for biochemical assay used to determine thiol content of protein in native and irradiated BSA. SDS-PAGE profile and CD spectroscopy studies were performed using working concentrations of BSA of 4.5 and 1 μM, respectively. 2.3. Gamma Irradiation procedure The BSA solutions were prepared in borosilicate glass vials and irradiated at two doses, 500 Gy and 1000 Gy (dose rate, 1.1 k Gy/h) using 60Co gamma ray irradiator, Model GC 5000, BRIT, Mumbai, India,). Protein samples containing 15 µM, 150 µM and 1500 µM EP was irradiated at both 500 Gy and 1000 Gy.
2.4. SDS-PAGE All the protein samples were run and analyzed using SDS-PAGE as suggested by Laemmli with some modifications [34]. 10 μl of each of the protein samples were added to 10 μl of 6.25 mM Tris HCL buffer (pH 6.8) containing 25% glycerol, 2% w/v SDS, 0.01% w/v bromophenol blue. These samples were heated for 5 mins at 95 °C and cooled in air. 20 μl of each of protein samples were loaded in the respective wells on an SDS-PAGE gel containing separating gel with 10% polyacrylamide and stacking gel with 4% polyacrylamide. The samples were electrophoresed at a constant voltage for 90 minutes (stacking at 45 V and resolving at 70 V). The gel was stained with 0.25% w/v Coomassie Brilliant Blue R 250 in water/methanol/acetic acid (9:9:2 by volume) at 25 °C with gentle shaking and destained with the destaining solution consisting of water/methanol/acetic acid (9:9:2 by volume). Gel image was captured using Gel DOC system (Biorad, USA). 2.5. UV-Vis Absorption measurement The UV-Vis absorbance measurement of protein samples (native, irradiated, pretreated with EP and irradiated) exposed to different doses that is 500 Gy and 1000 Gy was performed using UV-Vis spectrophotometer (Cary 100 BIO, Varian, Australia) equipped with a peltier thermostatic cell holder. The absorption of protein samples was measured using PBS as a reference. 3 ml of the each protein samples were kept in the 3.5 ml capacity quartz cuvette of 10 mm path length. All the samples were scanned from 200 nm - 400 nm wavelength range at 25 °C. 2.6. Fluorescence measurement Tryptophan oxidation and bityrosine formation in the protein samples were measured using spectrofluorophotometer (FS920, Edinburg instruments, Edinburg, UK). The excitation source was xenon lamp (450 W) and sample chamber was equipped with Peltier accessory. Scanning parameters for all measurements were optimized with slit width 4 nm for
excitation and emission, dwell time 0.2 s and wavelength step 0.5 nm. The measurements of tryptophan loss and bityrosine production in BSA samples were measured by keeping the protein sample (75 μM) in a 3.5 ml capacity quartz cuvette with a path length of 10 mm. The excitation wavelength was kept at 295 nm to excite selectively the tryptophan residues and emission was measured from 310 nm – 400 nm. Dityrosine content was measured in the protein samples at excitation wavelength of 325 nm and emission wavelength of 390 nm – 500 nm [35]. All the fluorescence measurements were performed at 25 °C. 2.7. Circular dichroism measurement The circular dichroism measurement was performed on spectropolarimeter (J18, Jasco, Japan) equipped with peltier accessory. The measurements were performed by the method given by Pathak et al [31]. The scan of each protein sample was taken in the wavelength range of 200 nm - 250 nm. CD melting experiment was performed on JASCO 815 spectropolarimeter interfaced with an IBM PC compatible computer, calibrated with D-Camphor Sulphonic acid. Samples were prepared by heating at 95 °C followed by gradual cooling to 25 °C. Melting data was collected at the ramp rate of 1 °C /min between the temperature ranges of 40 °C – 95 °C. Band width and data pitch were fixed to 1 nm and 0.2°C, respectively. CD melting data of native, irradiated and pretreated with EP and irradiated BSA samples to different doses of gamma radiation that is, 500 Gy and 1000 Gy, was recorded at the wavelength of 222 nm. CD measurements of all the samples were measured at 25 °C. 2.8. Protein carbonylation and DTNB assay Protein carbonyl formation of protein samples were evaluated using the methods suggested by Hawkins et al [36] with some modifications. The protein whether in the native or in irradiated without and with the treatment of EP (15 μΜ, 150 μΜ and 1500 μΜ) were incubated with equal volume of 10 mM DNPH (prepared in 2N HCl) in dark at 25 °C for 1
hour. The samples were precipitated by adding ice cold 50% TCA solution and centrifuged at 5000 rpm for 10 minutes. The pellets were washed 3 times with ethanol/ethyl acetate (1/1; v/v) solution to remove any unbound DNPH. The resulting pellet was resuspended in 1 ml 6M GuHCL. The protein samples were quantified for protein carbonylation formation at 371 nm against GuHCL as reference. The calculations were carried out according to the method given by Mishra et al [1]. In the DTNB assay, the protein samples were analyzed for the loss of thiol group in the irradiated BSA samples in the presence and absence of different concentrations of EP (15 μΜ, 150 μΜ and 1500 μΜ) by the method suggested by Hawkins et al [36] with some modifications. 1.5 ml of protein samples were mixed with 200 μl of 2 mM DTNB prepared in PBS (pH 7.4). The samples were kept for incubation period in dark for 30 minutes at 25 °C. The samples were analyzed for thiol content at 412 nm with PBS as reference using the molar extinction co-efficient of 13600 M-1 cm-1 [1]. 2.9. Hydroxyl radical scavenging assay Hydroxyl radical scavenging potential of EP was examined using 2-deoxy-D-ribose degradation assay and measured using a UV Vis spectrophotometer. 2-deoxy-D-ribose samples were prepared in 10 mM PBS, mixed with different concentrations of EP (15 μΜ, 150 μΜ and 1500 μΜ) and irradiated at radiation doses of 500 Gy and 1000 Gy. Each sample was mixed with 1 ml each of 10% TCA and 1% TBA (prepared in 0.05N NaOH) and heated at 95 oC for 30 minutes. Samples were cooled and absorbance was taken at 532 nm. 2.10. Statistical analysis The results were expressed as mean with standard error of mean (mean± SEM). The results were examined statistically by one way ANOVA with Tukey’s post hoc test analysis as
statistically significant at 95% confidence level (p<0.05) with the help of Graph Pad Prism version-5.0. 3. Results and discussion 3.1. SDS-PAGE Analysis It can be observed that the major band of native BSA was observed at 65 kDa in SDSPAGE of protein using protein markers of molecular weight ranging from 192-15 kDa (Fig. 2). The intensity of the major band of BSA was prominent in the native BSA sample, but at 500 Gy the band intensity is decreased which ultimately reduces significantly to a very low level at 1000 Gy with the lightening and light smearing of bands. Earlier, Mishra et al [1], Lee et al [9], Moon and Song [13] have shown that γ-irradiation of protein at lower doses of up to 1000 Gy causes the breakage of polypeptide chain that results in decrease of major protein band intensity with same amount of protein loaded. The various studies have shown that the loss of intensity of the major band and smearing of protein may be caused due to the degradation of polypeptide chains of protein at low radiation doses. Therefore, in the present work it is inferred that radiation exposure of 500 Gy and 1000 Gy caused significant damage in BSA. However, BSA samples pre-treated with different concentrations of EP (D1: 15 µM, D2: 150 µM, D3: 1500 µM) showed no appreciable change in the band intensity upon irradiation at 500 Gy and 1000 Gy (Fig. 2). It was observed that intensity of the major bands (65kDa) was retained as close to the intensity of native BSA in the presence of three different concentrations of EP. The observations of SDS-PAGE profile of BSA were found to be in accordance with earlier reported studies. Mishra et al [1] has showed that ferulic acid prevents the γ-irradiation induced damage of BSA by scavenging the free radicals. Similarly, Moon and Song [13] have also confirmed the protective effect of ascorbic acid against γ-radiation induced albumin damage by scavenging the reactive oxygen species. Therefore, SDS-PAGE profile of native, irradiated and pretreated with EP BSA irradiated at
different doses, 500 Gy and 1000 Gy confirmed the radioprotective effect of EP against gamma irradiation induced protein damage. 3.2. UV-Vis Spectroscopy UV-Vis spectroscopy is the most widely used biophysical technique for measuring changes in the absorbance of a molecule on binding and for assessing the damage [37]. In this study, this technique is used to determine the extent of damage produced by gamma radiation in DNA and the effect of EP in preventing that damage. In Fig. 3 (a and b), the UV- spectra of BSA shows an increase in the absorption intensity of BSA at high dose of gamma radiation, i.e., 1000 Gy but no change in the absorption intensity at low gamma radiation dose (500 Gy). There was a pioneering study which has recorded changes in the spectroscopic signals of BSA when irradiated to different doses of ionizing radiation. In the same work, the author has confirmed that there were a change in UV absorbance of BSA due to the breakage of covalent bonds of protein and disordering the protein’s structure [12]. Similar observations were recorded in the present study when BSA samples were irradiated to 500 Gy and 1000 Gy. While, the absorbance of BSA samples were retained when it was treated with different concentrations of EP prior γirradiation at 500 Gy and 1000 Gy. It was occurred because the ordered structure of BSA protein was retained in the presence of EP as it binds to BSA significantly [31]. 3.3. Fluorescence measurements 3.3.1 Fluorescence bityrosine formation When the BSA protein was irradiated with gamma radiation both at 500 Gy (Fig. 4a) and 1000 Gy (Fig. 4b), it leads to increase in the fluorescence emission intensity of protein at 425 nm. The increase in fluorescence intensity around 425 nm, in case of ionizing irradiation induced structural modification of serum protein, was due to the formation of tyrosine dimers formed by reaction of tyrosine residues of protein with water radiolysis
products, mainly ˙OH [35]. This ˙OH abstracts the hydrogen from tyrosine residues forming tyrosyl radicals which in turn reacts with other tyrosyl radicals and forms bityrosine and cross-links the protein that resulted in increase in fluorescence intensity of protein [3, 38, 39]. However, when BSA was irradiated in the presence of different concentrations of EP, the fluorescence intensity at 425 nm decreased steadily with the increase in EP concentration. The observation suggested the inhibition of bityrosine formation in the presence of EP. 3.3.2 Fluorescence tryptophan degradation The fluorescence intensity at emission wavelength ranging 339 nm - 350 nm is due to the emission from tryptophan residues when excited selectively at 295 nm [37, 40]. Therefore, fluorescence band around 339 nm is characteristic marker for tryptophan residues in BSA. The decrease in fluorescence intensity of BSA around 339 nm indicated that irradiation has damaged the tryptophan residues due to the oxidation by the ROS formed by the radiolysis of water [35]. The fluorescence emission of BSA around 339 nm decreases significantly as protein samples were irradiated with radiation doses of 500 Gy (Fig. 5a) and 1000 Gy (Fig. 5b). Fluorescence quenching of protein increases proportionally with gamma radiation doses [12, 13]. Similar to the observation in case of bityrosine formation, EP reduces the impact of gamma irradiation by retaining the tryptophan emission in protein samples when administered with EP. Besides, EP shows its protective effect in proportion to its concentration. 3.4. CD measurement CD is an extensively sensitive technique to investigate the conformational changes in protein. Different structural element of protein such as α- helix, β-sheet, random coils and turns all have characteristic CD signatures. The proteins containing α- helical content exhibit two negative CD peaks in UV region centered on 208 nm and 222 nm while having β-content shows negative band at 218 nm [41]. The negative peak centered at 208 nm arises
due to the exciton splitting of the peptide * transition whereas the peak at 220 nm originates due to n* transition [42, 43]. A CD study is performed in the wavelength range from 200 nm to 250 nm in absence as well as in presence of radiation and EP. Fig. 6 demonstrates the typical Far-UV CD spectra of BSA in the absence and presence of various gyration of radiation and EP. BSA displayed a typical CD signature with negative ellipticity centered at 208 nm and 222 nm having considerable amount of alpha helical content. In presence of EP the helicity of protein gets increased with little shifts from 208 nm peak to the 211 nm. This shows that EP interacts with BSA and stabilizes the protein by binding to it. When BSA protein alone is irradiated with gamma radiation in 500 Gy as well as 1000 Gy the helicity of protein diminishes to a greater extent. The protein structure collapses at 1000 Gy dose. A considerable reduction in magnitude of negative ellipticity of the CD peaks is observed at both wavelengths. It is clearly observed from CD spectra that peak at 208 nm and 222 nm both decrease sharply but the decrease in ellipticity was observed largely at 208 nm peak. However, after addition of EP in BSA prior gamma irradiation with 500 Gy gamma radiation, the helicity of protein is retained to a larger extent while in 1000 Gy irradiated BSA with prior addition of EP, retention of protein helicity is less. It is clearly seen from CD spectra that in presence of EP the protein retains its structure and EP protects the secondary structure of protein. Radiation alters the protein conformation and disrupts the intramolecular bonding between amino acids in protein. It is also observed from the CD studies that EP can help in retaining the conformation and protecting the secondary structure of protein from radiation damage. Further the stability was investigated by CD melting studies on BSA protein in presence of radiation and EP. Thermal stability of BSA protein is investigated by CD melting studies. CD melting experiment was performed in the range of 200 nm to 250 nm by heating at the rate of 0.5 ᵒC
in the temperature range of 40 ᵒC -95 ᵒC. CD melting spectra is displayed in Fig. 7 in the absence and presence of gamma radiation and EP. CD melting spectra clearly demonstrates that BSA protein alone show melting temperature (Tm) at approximately 55 ᵒC whereas on addition of EP, the Tm is observed somewhere around 75 ᵒC. So, it is clearly observed that EP predominantly stabilizes the protein secondary structure. Further BSA was irradiated at different doses of gamma radiation i.e. with 500 Gy and 1000 Gy. Thermal stability is again observed and it is seen from spectra that in the presence of radiation, protein structure collapses and all the factors like intramolecular forces and hydrogen bonding are disrupted due to interaction with gamma radiation. Damage is observed to a greater extent in the presence of 1000 Gy dose of radiation. It was reported in literature that unfolding of protein proceeds in various steps including native to extended and followed by unfolding. In these steps protein retain its natural folded state to some extent while in unfolding state protein starts to melt [44-46]. Further on the addition of EP in irradiated BSA, stability of protein structure is observed and it was shown by CD melting spectra that EP provides the retention of protein structure and the protein regains its conformation again. This study is in well agreement with the CD studies where the protein secondary structure is well retained in presence of EP at different doses of gamma radiation. These studies can help in understanding the interaction of various drugs with BSA protein and doses of radiation which can cause less alteration to the protein conformation. These types of studies play a pivotal role in biomedical field. 3.5. Protein carbonylation and DTNB Assay Carbonylation is the stable and irreversible transformation that damages the protein and hence its function [4, 47]. Ionizing radiation enhances the protein oxidation and causes the formation of carbonyl groups in the proteins [1]. Protein carbonyls are generated by
multiple radicals directly and by singlet oxygen species as a result of secondary reactions [36]. In Fig. 8, it is shown that the carbonyl content in BSA in the presence of two doses of gamma radiation, 500 Gy and 1000 Gy, has increased from 14.23 μM to 71.7 μM and 82.67 μM, respectively. In the presence of different concentrations of EP [15 µM (D1), 150 µM (D2) and 1500 µM (D3)], the formation of carbonyl content in BSA reduces at both the radiation doses. Mishra et al [1] has also showed in their study that the ferulic acid prevented the formation of carbonyls in the BSA after gamma irradiation. This study is in agreement with the earlier performed study and hence suggests the protective effect of EP on gamma radiation induced damage in BSA. Most of the proteins including BSA contain thiol groups as cysteine residues and oxidation of these thiol groups is an indicator of protein modification. The modification of these cysteine residues can be persuaded by many oxidants like peroxides, peroxynitrile, singlet oxygen and many radicals that can form multiple products such as cystine, mixed disulfides, etc. [36]. The loss of thiol groups from the proteins is related to loss of their activity [1]. When DTNB reacts with reduced thiols, it results in formation of 5-thio-2-nitrobenzoic acid (TNB). Gamma irradiation of BSA at 500 Gy caused loss of thiol content from 62.2 μM to 40 μM. Irradiation to 1000 Gy caused no further change in the thiol content suggesting that protein damage in terms of thiol content reached saturation at 500 Gy. But in the presence of increasing doses of EP, the loss of thiol content reduced proportionally to the presence of EP in the protein sample (Fig. 9). Hence, the assay confirmed the protective effect of EP against radiation induced thiol oxidation in protein. 3.6. Hydroxyl radical scavenging assay
Malondialdehyde (MDA) is a TBA-reactive matter that is produced by free-radical mediated destruction of carbohydrate moieties, e.g., 2-D-deoxyribose sugar, especially by hydroxyl radicals [48]. The ionizing radiations cause the hydrolysis of water that leads to the generation of hydroxyl radicals leading to DNA damage [3]. For determining the hydroxyl radical scavenging ability of EP, 2-D-deoxyribose damage was produced by hydroxyl radicals generated by ionizing radiations and examined at different concentrations of EP (125 µM to 4000 µM). A radiation dose of 500 Gy and 1000 Gy produced around 2.95
µM
and
3.82
µM
of
malondialdehyde
(MDA)
(Fig.
10).
The results suggest that EP decreased the formation of MDA in a concentration-dependent manner in samples irradiated with 500 Gy and 1000 Gy. EP at 4000 µM decreased the hydroxyl radical mediated formation of MDA by 80% and 68% at 500 Gy and 1000 Gy, respectively. 4. Conclusion The anti-radical property of EP was assessed against gamma radiation induced BSA damage through
SDS-PAGE,
spectroscopic
techniques
including
UV-Vis
spectroscopy,
fluorescence spectroscopy and CD spectroscopy and biochemical analysis such as, carbonyls formation, thiols oxidation and malondialdehyde formation in proteins. All these above studies show that when BSA is irradiated with gamma radiation in the presence of EP, the damage in BSA protein is prevented due to its radical scavenging property. Hence, EP has a therapeutic effect in protecting protein against gamma radiation and can be proposed as a better radioprotective agent. With further analysis involving in vivo and in vitro safety studies, an effective formulation either oral or topical can be prepared. These formulations can be used as prophylactic and therapeutic agents for the treatment of injuries involving radiation exposure.
Conflict of Interest There is no conflict of interest among the authors. Acknowledgements The authors are sincerely thankful to Director INMAS, DRDO for supporting and making available the facilities to carry out the research work. Authors are sincerely thankful to Head of department, Department of Chemistry, University of Delhi and Principal, Miranda House, for providing spectroscopic techniques to carry out the research. One of the authors Ms Deepti Sharma thanks UGC for providing senior research fellowship. Ms Lajpreet Kaur is thankful to CSIR for providing junior research fellowship. Figure legends Fig. 1 Molecular structure of ethyl pyruvate Fig. 2 SDS-PAGE profile of native and gamma-irradiated BSA at two doses: 500 Gy and 1000 Gy in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM]. SDS-PAGE is showing smear and almost a significant loss of BSA when irradiated at 1000 Gy, retaining the protein in the presence of EP. Similarly, at 500 Gy, the damage was low as smeared product was less but gets retained in the presence of EP. Fig. 3 a) UV-Vis spectra of irradiated BSA at (a)500 Gy and (b) 1000 Gy in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM]. The spectra indicated the negligible change in absorbance of irradiated BSA in the presence of higher concentrations of EP. Fig. 4 Fluorescence spectra of native and irradiated BSA at (a) 500 Gy; (b) 1000 Gy in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM] preventing bityrosine formation within BSA protein.
Fig. 5 Fluorescence spectra of native and irradiated BSA (a) at 500 Gy; (b) at 1000 Gy in the absence and presence of different concentrations of EP [D1: 15 µM EP; D2: 150 µM EP and D3: 1500 µM EP] preventing tryptophan degradation within BSA protein. The fluorescence intensity of tryptophan in irradiated BSA sample was found negligibly changed with the addition of EP. Fig. 6 CD spectra of native and irradiated BSA. BSA was irradiated with gamma radiation at 500 Gy and 1000 Gy in the absence and presence of EP (D3: 1500 µM) showing that EP helps in retention of protein conformation in the irradiated BSA. Fig. 7 CD melting spectra of native nd gamma irradiated BSA (500 Gy and 1000 Gy) in the presence of highest concentration of EP (D3: 1500 µM). In the presence of EP, the irradiated BSA protein retained its original Tm. Fig. 8 The graph showing the formation of carbonyls in BSA when irradiated with gamma radiation (500 Gy and 1000 Gy) in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM] (p<0.05). In the presence of EP, the concentration of carbonyls is decreasing significantly with the increasing concentration of EP. Fig. 9 The graph showing the content of thiols in BSA when irradiated with gamma radiation (500 Gy and 1000 Gy) in the absence and presence of different concentrations of EP [D1: 15 µM; D2: 150 µM and D3: 1500 µM] (p<0.05). The thiol content of irradiated BSA is increasing significantly with the increasing concentration of EP. Fig. 10 The graph showing the malondialdehyde content in BSA when irradiated with gamma radiation (500 Gy and 1000 Gy) in the absence and presence of different concentrations of EP [E1: 125 µM; E2: 250 µM; E3: 500 µM; E4: 1000 µM; E5: 2000 µM; E6: 4000 µM]
(p<0.05). The malondialdehyde which is a marker of oxidative damage decreasing significantly with the increasing concentration of EP.
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Protection by Ethyl Pyruvate against Gamma Radiation Induced Damage in Bovine Serum Albumin Deepti Sharmaa, Anju Singhb, Shrikant Kukretib, Mallika Pathakc, Lajpreet Kaura, Vinod Kaushika, Himanshu Ojhaa* a
Protection and Decontamination research group, Division of CBRN Defence, Institute of Nuclear Medicine and Allied Sciences, Delhi-110054
b
c
Nucleic Acid Research Lab, Department of Chemistry, University of Delhi, Delhi-110007
Department of Chemistry, Miranda House, University of Delhi, Delhi-110007
*Corresponding author Dr Himanshu Ojha Scientist E Protection and Decontamination research group Division of CBRN Defence Institute of Nuclear Medicine and Allied Sciences Timarpur, Delhi 110054 Tel +91-11-23905186 Fax+91-11-23919509 Email: [email protected]
O O
O Fig. 1
kDa
1
2
3
4
5
6
7
195
102 65
41
Fig. 2
8
9
10
11
12
13
a
b
Fig. 3
a
b
Fig. 4
Fig. 5
200
210
220
230
240
250
10 0 -10
θ deg cm2 mol-1
-20 -30
-40
BSA Control BSA +D3
-50
Irrad BSA 500
-60
Irrad BSA 1000 IrraBSA+D3 500
-70
IrraBSA+D3 1000
-80 -90
Wavelength (nm) Fig. 6
-10 40
50
60
70
80
90
100
-20
Θ deg cm2 mol-1
-30
-40
-50 BSA Control
-60
BSA +D3 Irr BSA 500
-70
-80
Irr BSA 1000
Temperature °C
Fig. 7
Irr BSA 500 +D3 Irr BSA 1000 +D3
Fig. 8
Fig.9
Fig. 10
Highlights
Radioprotective effect of ethyl pyruvate (EP) on bovine serum albumin was studied.
EP prevented bityrosine formation & tryptophan degradation.
SDS-PAGE showed low BSA protein damage in presence of EP.
Biochemical assays proved protective role of EP on protein (BSA) damage.