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The effect of fullerenol C60(OH)~30 on the alcohol dehydrogenase activity irradiated with X-rays
Radiation Physics and Chemistry 97 (2014) 102–106
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Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Short Communication
The effect of fullerenol C60(OH) 30 on the alcohol dehydrogenase activity irradiated with X-rays Anita Krokosz a,n, Jacek Grebowski a, Aleksandra Rodacka a, Beata Pasternak b, Mieczyslaw Puchala a a b
Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland Laboratory of Molecular Spectroscopy, Faculty of Chemistry, University of Lodz, Lodz, Poland
H I G H L I G H T S
Fullerenol C60(OH) 30 itself do not modify the activity of alcohol dehydrogenase (ADH). Fullerenol protects ADH against radiation inactivation due to simple competition for the dOH radicals. Fullerenol has the reduced ability to prevent the formation of protein peroxides. Since the main role in radiosensitivity of ADH is played by –SH groups, fullerenol could prevent oxidation of –SH groups by electrostatic interactions.
art ic l e i nf o
a b s t r a c t
Article history: Received 7 May 2013 Accepted 6 November 2013 Available online 17 November 2013
In the present study the effect of X-irradiation on the alcohol dehydrogenase (ADH) activity in the presence of nanoparticles of fullerenol C60(OH) 30 under aerobic conditions was investigated in order to assess the potential radioprotective properties of fullerenol. Fullerenol at 75 mg/mL decreased the radiation yield of inactivation of ADH irradiated with fullerenol by 20% comparing to ADH irradiated without fullerenol. Under conditions used during irradiation, 50% of d OH radicals could react with fullerenol and 50% could react with ADH. Thus, it can be assumed that protective effect of fullerenol on the radiation inactivation of ADH was mostly due to scavenging dOH radicals by fullerenol. Moreover, fullerenol did not protect against post-irradiation damage as the Ginact for ADH irradiated with fullerenol was still 20% lower than for ADH irradiated without fullerenol after 24 h from irradiation. Additionally, fullerenol at 75 mg/L had no influence on the activity of unirradiated ADH up to 24 h. We concluded that fullerenol C60(OH) 30 protected ADH against radiation inactivation due to simple competition for the dOH radicals and did not modify its activity by association with the protein as it was proved in our previous papers for erythrocyte membrane proteins. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Fullerenol Alcohol dehydrogenase X-radiation
1. Introduction Fullerenes are chemical structures made of carbon atoms. The stable form of fullerene is a molecule composed of 60 carbon atoms arranged in a soccer ball-shaped structure. Fullerene C60 has 30 double bonds (60 π electrons) which determine the excellent electron
Abbreviations: ADH, alcohol dehydrogenase from baker’s yeast; FullOH, fullerenol C60(OH) 30; LQ, linear-quadratic model; D37, the irradiation dose (in Gy) at which the enzyme activity decreased to 37% of the initial activity n Correspondence to: Division of Radiobiology, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/ 143 Pomorska Street, 90-236 Lodz, Poland. Tel.: þ 48 42 635 44 57; fax: þ48 42 635 44 73. E-mail addresses: [email protected], [email protected] (A. Krokosz). 0969-806X/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radphyschem.2013.11.009
donor or acceptor capabilities of fullerenes. Because of their specific structure, fullerenes can be effective radical scavengers or prooxidants (Grebowski and Krokosz, 2010; Ratnikova et al., 2011; Kong and Zepp, 2012). Chemical modification of the fullerene carbon cage by an attachment of hydroxyl groups can be an effective way to overcome the low solubility of fullerenes in aqueous solutions. Due to the presence of hydroxyl groups on the surface of fullerenol molecules, it is possible for them to take part in many interactions, e.g. creating hydrogen bonds with biomolecules (Grebowski et al., 2013a, 2013b; Bhattacharya et al., 2012). Hydrogen bonds play an important role due to their frequent occurrence in biological systems and their role in biochemistry (Schuster and Wolschann, 1999; Krych and Gebicka, 2013; Moosavi-Movahedi et al., 2013). Ionizing radiation is known to cause irreversible alterations of protein conformation and function (Riley, 1994; Du and Gebicki, 2004;
A. Krokosz et al. / Radiation Physics and Chemistry 97 (2014) 102–106
Rodacka et al., 2012). Protein damage due to indirect effects of irradiation could be minimized by optimization of the irradiation conditions e.g. by the use of antioxidants. Therefore the search for new, efficient radioprotectors is needed (Saloua et al., 2011). Due to their hydrophilic properties and the ability to scavenge free radicals, fullerenols may provide a serious alternative to the currently used pharmacological methods in chemotherapy, treatment of neurodegenerative diseases, and radiobiology. Additionally, due to the hollow spherical shape, fullerenols may be used as drug carriers (Grebowski et al., 2013c). The results obtained in many laboratories show that polyhydroxylated fullerenes, aka fullerenols or fullerols C60(OH)n, with more than 20 hydroxyl groups attached to carbon cage are watersoluble and exert mainly antioxidant/cytoprotective activity in biological systems (Cai et al., 2009; Vávrová et al., 2012) and may protect biomolecules and cells against ionizing radiation (Grebowski and Krokosz, 2010; Zhao et al., 2005; Bogdanović et al., 2008). However, there are a few studies regarding the toxic effects of fullerenol (Su et al., 2010; Wielgus et al., 2010). As irradiation of enzymatic proteins leads to their inactivation, we investigated the potential protective effect of fullerenol C60(OH) 30 against X-radiation on alcohol dehydrogenase and the effect of post-irradiation incubation in the presence or absence of fullerenol under aerobic conditions.
2. Experimental Fullerene C60 (99.5%) was purchased from SES Research (Houston, TX, USA). Alcohol dehydrogenase (ADH) (alcohol:NADþoxidoreductase, EC 1.1.1.1), molecular weight of 147 kDa from baker's yeast was purchased from Sigma-Aldrich (Poznan, Poland). All other chemicals were of analytical grade and were purchased from SigmaAldrich (Poznan, Poland) or POCH (Gliwice, Poland). All solutions were made with water purified by the Milli-Q system. 2.1. Synthesis of fullerenol C60(OH) 30 Polyhydroxylated fullerene derivative C60(OH) 30 was obtained by a method of Wang et al. (2005), with some modifications. Fullerene C60 and solid NaOH, in the presence of 30% H2O2 were ground in a glass mortar at a room temperature for 25 min until the color of the mixture turned to yellow-brown. Then, the incubation in a waterbath at 60 1C was carried out for 20 min. Deionized water was added to the reaction mixture and it was stirred for 24 h to dissolve the sludge. The solution of fullerenol was separated from any insoluble blend by centrifugation at 7000 g for 15 min (20 1C). After precipitation of fullerenol by methanol and resolubilization in deionised water, traces of NaOH were removed by ion-exchange chromatography with Amberlit MB-20. The structure of the obtained hydroxyl derivative of fullerene C60 was confirmed by IR spectrophotometry (NEXUS FT-IR spectrometer), 1H-NMR (Varian Gemini 200 MHz), 13C-NMR (Bruker Avance III 600 MHz) and mass spectroscopy MS-ESI (Varian 500 MS).
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enzyme was measured after 1 h from the end of irradiation or after 8 and 24 h of incubation at 25 1C in the dark. 2.3. The determination of enzyme activity Concentration of the ADH was determined on the basis of the absorbance measurements at 280 nm using the absorption coefficient of E1% 280 ¼ 14:6. ADH activity was measured by the method of Bonnichsen and Brink (1955). The activitiy of the enzyme was determined on the basis of the rate of reduction of NAD þ to NADH. The formation of NADH was estimated by the measurement of absorbance at λ ¼ 340 nm. Spectrophotometric measurements were carried out at room temperature in a CARY-1 apparatus (Varian, Melbourne, Australia). The resulting data were fitted by the linear or the linear-quadratic (LQ) model using Excel 2010 software (Microsoft Sp. z o.o., Warsaw, Poland). The doses corresponding to 37% of the enzyme activity (D37) and α, β values calculated from the fitted models were analyzed to compare the effect of fullerenol C60(OH) 30. Three independent experiments were performed for the each sample. 2.4. Calculations The radiation yield of Ginact for enzymes irradiated was calculated on the basis of the value of the dose D37 according to the formula cADH mol Ginact ¼ ρD37 J where cADH is the enzyme concentration in mol dm 3, D37 the irradiation dose (in Gy) at which the enzyme activity decreased to 37% of the initial activity and ρ the density of solution about 1 kg dm 3 (Rodacka et al., 2010). The radiation yield of dOH radicals reacting with ADH in the presence of fullerenol C60(OH) 30 was determined as described previously (Kowalczyk et al., 2008). Briefly, the radiation yield of d OH radicals reacting with enzyme in the presence of fullerenol was determined using the equation: G¼
kðOH þ EÞ ½E G0 ¼ ω G0 kðOH þ EÞ ½E þ kðOH þ FullOHÞ ½FullOH
where G0 – the yield of dOH radicals in the absence of fullerenol; in the atmosphere of air it was accepted that G0 ¼ 2.82 10-7 mol J-1; [FullOH] – the concentration of fullerenol; k – the appropriate rate constants of reaction of dOH radicals with enzyme [E] or fullerenol [FullOH]; for fullerenol - rate constant was 2.0 109 dm3 mol-1 s-1 (unpublished results); for ADH – rate constant was 1.8 1011 dm3 mol-1 s-1 (Buxton et al., 1988). ω – the fraction of dOH radicals reacting with enzyme in the competition with FullOH. If only the reaction of dOH radicals with enzyme was responsible for the expected inactivation, the yield would be calculated on the basis of the following equation: Gexp: inactiv: ¼
2.2. Conditions of irradiation
G 0 G ¼ ωG0inactiv: G0 inactiv:
where The ADH was dissolved in 0.02 M potassium phosphate buffer, pH 7.4, at the concentration of 0.1 mgprotein/mL (6.8 10-7 mol dm-3) and irradiated in the presence of or without fullerenol (FullOH) (75 mg/L). Solutions were irradiated in air using Stabilipan 2 an X-ray machine (Siemens, Germany) 195 kV, 18 mA, with an aluminum filter (2 mm). The dose rate estimated with a Fricke dosimeter was 4.1 Gy/min. Irradiation of samples was performed in a glass beaker with gentle mixing with magnetic bar. The activity of the
G0inactiv: – the yield of inactivation of the enzyme irradiated in the absence of fullerenol; Gexp: inactiv: – the expected yield of inactivation of the enzyme irradiated in the presence of fullerenol. It was assumed that molecular mass of fullerenol C60(OH) 30 was 1230 D and molar concentration 6.1 10-5 mol dm-3.
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2.5. Statistical analysis
log(%ActADH)= -(5.74x10-3D+3.86x10-5D2)+2
2.00
log (% of ADH activity)
The experimental values are expressed as the mean and the error bars indicate the standard error of the mean (SEM) calculated for each data point. All experiments were run 3–7 times. Significant differences between data were assessed by the paired Student's t-test with p o0.05. 3. Results and discussion
R2 = 0.9999 1.50
R2 = 0.9994 0.50 ADH
0.00
2.00
log(%ActADH) =-7.54x10-3D+2
log (% of ADH activity)
R2 = 0.9988 1.50
log(%ActADH) =-9.48x10-3D+2 R2 = 0.9966
0.50 ADH
0.00
0
ADH+fullerenol
20
40
60
80
100
Dose [Gy] Fig. 1. The effect of X-irradiation on the enzyme (alcohol dehydrogenase) irradiated in the absence or presence of fullerenol (75 mg/L) under air as the dependence of the logarithm of relative ADH activity on the radiation dose. Measurements were done 1 h after the end of irradiation. Each point is the mean of 3–7 independent experiments 7 SEM.
0
ADH+fullerenol
20
40
60
80
100
Dose [Gy]
2.00
log (% of ADH activity)
Alcohol dehydrogenase from baker's yeasts contains 2 cysteine residues (Cys-43, Cys-153) and 1 histidine residue (His-66) complexed with a Zn atom in the active site. ADH is effectively inactivated by radiation-induced dOH radicals (Kowalczyk et al., 2008; Rodacka et al., 2010). A significant role for inactivation susceptibility is played by the closest neighborhood of the active site; among others, by the number of more reactive amino acid residues such as Cys, Trp, Phe, His and Met. At the distance of 10 Å from the active site of ADH, there are 12 sensitive residues including 4 sulfur residues (PDB, code 2HCY). The results of ADH inactivation by X-radiation in the presence or absence of fullerenol are shown in Fig. 1. The dependence of log (% activity) on the radiation dose is linear, as it was obtained in the previous work (Kowalczyk et al., 2008). The presence of fullerenol at 75 mg/L did not change the character of the dependence but decreased the slope of the line (α) from 9.48 to 7.54 Gy 1. The slope of the line (inactivation coefficient) characterize radiosensitivity of the enzyme. It indicated that fullerenol could protect ADH with the efficiency of 20.5%. The post-irradiation incubation of the enzyme enhanced the inactivation of ADH as a result of previous irradiation (Fig. 2) and the linear-quadratic model (LQ) was used to describe the inactivation of ADH after 8 h and 24 h from the irradiation. The parameters calculated from the semi-logarithmic dose-effect curves (Fig. 2) for the dependence of relative ADH activity on the radiation dose for enzyme irradiated with X-rays without or with fullerenol at the concentration of 75 mg/L and incubated at 25 1C for 8 and 24 h are gathered in Table 1. It is worth to underline, that the concentration of fullerenol used in our work was rather high (75 mg/L) so the observed effects were explicitly pronounced. Moreover, the fullerenol at 75 mg/L and the time of incubation had no influence on the activity of unirradiated ADH up to 24 h of incubation at 25 1C in the dark (data not shown).
1.00
log(%ActADH)=-(8.00x10-3D+4.75x10-5D2)+2
1.00
log(%ActADH) =-(5.83x10-3D+6.44x10-5D2)+2 R2 = 0.9993
1.50
1.00
log(%ActADH)=-(8.17x10-3D+8.20x10-5D2)+2 R2 = 0.9995
0.50 ADH
0.00
0
ADH+fullerenol
20
40
60
80
100
Dose [Gy] Fig. 2. The effect of incubation on the enzyme (alcohol dehydrogenase) irradiated in the absence or presence of fullerenol (75 mg/L) under air as the dependence of the logarithm of relative ADH activity on the radiation dose, (A) after 8 h incubation; (B) after 24 h incubation from the end of irradiation. Samples were incubated at 25 1C. Each point is the mean of 3–7 independent experiments 7 SEM.
Table 1 The parameters calculated from the dose-effect curves for the dependence of logarithm of relative ADH activity on the radiation dose for enzyme irradiated with X-radiation without or with fullerenol at the concentration of 75 μg/ml and incubated at 25 1C. Time of incubation (h)
8 24
α 103 (Gy 1)
β 105 (Gy 2)
ADH
ADH þfullerenol
ADH
ADH þfullerenol
8.00 8.17
5.74 5.83
4.75 8.20
3.86 6.44
The linear-quadratic equation is well suited to fit experimental survival curves (Brenner et al., 1998) but it describes the postirradiation inactivation of ADH, the enzyme of oxidoreductase class, in an excellent way as well. Based on the linear-quadratic model, we can assume that the limiting slope (α) describing the linear part of the dose-effect curve, describes direct damage to the active site of the enzyme leading to its inactivation. However, the coefficient (β) for the dose-squared term of the curve indicates the participation of indirect damage to the enzyme leading to the inactivation of ADH. The post-radiation incubation of ADH did not result in the change of the α coefficient. However, the β coefficient increased 1.7-fold with the time of incubation independently of the presence of fullerenol (Table 1). This observation indicated the increase of post-radiation damage resulting from the modification of the structure of the enzyme by molecular oxygen from the air. As it was shown by Rodacka et al. (2010), the main role in radiosensitivity of ADH is played by -SH groups. Oxygen could oxidize –SH groups to disulfides in neutral pH (Bagiyan et al., 2003). The initial modifications in ADH structure by radiation could additionally facilitate oxidation of –SH groups by molecular oxygen. From the obtained in Figs. 1 and 2 dependences D37 values were determined, which served as a basis for the calculation of the
A. Krokosz et al. / Radiation Physics and Chemistry 97 (2014) 102–106
Table 2 Doses D37 (the dose after which 37% of initial activity remains) and the radiation yields (Ginact) of inactivation of ADH irradiated with X-radiation without or with fullerenol at the concentration of 75 μg/ml and incubated at 25 1C; mean value7 SEM; significant difference compared to ADH (without fullerenol) is indicated by asterisk at po 0.05. Time of incubation (h)
D37 [Gy] ADH
Ginact [nmol/J] ADHþ fullerenol ADH
Without incubation 45.5 7 2.7 57.3 7 2.5n 8 43.0 7 6.3 54.9 76.7n 24 38.2 7 1.9 48.37 3.4n
ADHþ fullerenol
14.9 7 0.9 11.9 7 0.5n 15.8 7 2.4 12.4 7 1.6n 17.8 7 0.9 14.17 1.0n
radiation yield of inactivation according to the formula described in the Section 2.4. The radiation yield of inactivation of ADH irradiated with fullerenol was 20% lower than of ADH irradiated without fullerenol (Table 2). The efficiency of inactivation of ADH by radiation-generated d OH and O2 d radicals is 0.0350 and 0.0059, respectively (Rodacka et al., 2010) and we could take into attention mainly inactivation caused by dOH. In the present study, under conditions used during irradiation, 50% of dOH radicals could react with fullerenol and 50% could react with ADH as it was calculated according to the Section 2.4. Thus, it can be assumed that protective effect of fullerenol on the radiation inactivation of ADH was mostly due to scavenging d OH radicals by fullerenol. Guldi and Asmus, 1999, observed a negative correlation between the rate constant of scavenging dOH and the number of functional substituents in the range of different derivatives of fullerene C60. Hydroxyl radicals could abstract H atom or electron from the fullerenol as well as add to the double bonds. The more functional groups attached to the fullerene cage, the fewer number of π bonds in the molecule. This implies a decrease in possibility to scavenge free radicals and antioxidant capacity of fullerenol with increasing number of –OH groups attached to the carbon cage. However, our results revealed that highly hydroxylated fullerene exhibited an excellent solubility in water and kept their properties of scavenging dOH radicals simultaneously. The post-irradiation inactivation of the alcohol dehydrogenase irradiated in the absence or in the presence of fullerenol increased with the time of incubation (Table 2). However, fullerenol did not protect against post-irradiation damage as the Ginact for ADH irradiated with fullerenol was still 20% lower than for ADH irradiated without fullerenol after 24 h from irradiation. Fullerenol did not prevent the post-irradiation damage to the enzyme in our experimental system, which suggested the reduced ability of fullerenol to prevent the formation of protein peroxides. Protein peroxides form in high yields from proteins exposed to a wide range of ROS including hydroxyl radicals. Protein and peptide hydroperoxide groups have a half-life of several hours at room temperature and can inactivate enzymes. The first reaction product is a carbon-centered radical (Prd), rapidly converted in the presence of oxygen to a peroxyl radical (PrOOd) and finally to protein hydroperoxide (PrOOH). The precursor protein radicals can efficiently destroy antioxidants present in the experimental system (Du and Gebicki, 2004). Moreover, fullerenol could have associated with ADH by electrostatic, dipolar or Van der Waals interactions. The possibility of interaction of fullerenol with proteins was proved in our previous papers (Grebowski et al., 2013a, 2013b). Relative polar surface area of ADH is 0.375 so that one third of surface area of ADH which is 43577 Å (Rodacka et al., 2010) could interact by electrostatic and dipolar interactions with –OH groups of fullerenol. The interactions of proteins and nanoparticles (NPs) are reported in the newest papers considering protein corona
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(Fenoglio et al., 2011; Podila et al., 2012). These interactions influenced, among others, proteins activity, charge-transferinduced conformational changes and particles charge and reactivity. The ratio of the observed effectiveness of inactivation to the effectiveness of expected inactivation (t) was calculated according to the formula t¼
Ginactiv: obs: Gexp: inactiv:
The “t” parameter was 1.6 meaning that there was no or very little damage by secondary products of the reaction of the fullerenol with dOH radicals. We concluded that fullerenol C60(OH) 30 protected ADH against radiation inactivation due to simple competition for the d OH radicals and did not modify its activity by association with the protein as it was proved in our previous papers for erythrocyte membrane proteins (Grebowski et al., 2013a, 2013b).
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Contents lists available at ScienceDirect
Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Short Communication
The effect of fullerenol C60(OH) 30 on the alcohol dehydrogenase activity irradiated with X-rays Anita Krokosz a,n, Jacek Grebowski a, Aleksandra Rodacka a, Beata Pasternak b, Mieczyslaw Puchala a a b
Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland Laboratory of Molecular Spectroscopy, Faculty of Chemistry, University of Lodz, Lodz, Poland
H I G H L I G H T S
Fullerenol C60(OH) 30 itself do not modify the activity of alcohol dehydrogenase (ADH). Fullerenol protects ADH against radiation inactivation due to simple competition for the dOH radicals. Fullerenol has the reduced ability to prevent the formation of protein peroxides. Since the main role in radiosensitivity of ADH is played by –SH groups, fullerenol could prevent oxidation of –SH groups by electrostatic interactions.
art ic l e i nf o
a b s t r a c t
Article history: Received 7 May 2013 Accepted 6 November 2013 Available online 17 November 2013
In the present study the effect of X-irradiation on the alcohol dehydrogenase (ADH) activity in the presence of nanoparticles of fullerenol C60(OH) 30 under aerobic conditions was investigated in order to assess the potential radioprotective properties of fullerenol. Fullerenol at 75 mg/mL decreased the radiation yield of inactivation of ADH irradiated with fullerenol by 20% comparing to ADH irradiated without fullerenol. Under conditions used during irradiation, 50% of d OH radicals could react with fullerenol and 50% could react with ADH. Thus, it can be assumed that protective effect of fullerenol on the radiation inactivation of ADH was mostly due to scavenging dOH radicals by fullerenol. Moreover, fullerenol did not protect against post-irradiation damage as the Ginact for ADH irradiated with fullerenol was still 20% lower than for ADH irradiated without fullerenol after 24 h from irradiation. Additionally, fullerenol at 75 mg/L had no influence on the activity of unirradiated ADH up to 24 h. We concluded that fullerenol C60(OH) 30 protected ADH against radiation inactivation due to simple competition for the dOH radicals and did not modify its activity by association with the protein as it was proved in our previous papers for erythrocyte membrane proteins. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Fullerenol Alcohol dehydrogenase X-radiation
1. Introduction Fullerenes are chemical structures made of carbon atoms. The stable form of fullerene is a molecule composed of 60 carbon atoms arranged in a soccer ball-shaped structure. Fullerene C60 has 30 double bonds (60 π electrons) which determine the excellent electron
Abbreviations: ADH, alcohol dehydrogenase from baker’s yeast; FullOH, fullerenol C60(OH) 30; LQ, linear-quadratic model; D37, the irradiation dose (in Gy) at which the enzyme activity decreased to 37% of the initial activity n Correspondence to: Division of Radiobiology, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/ 143 Pomorska Street, 90-236 Lodz, Poland. Tel.: þ 48 42 635 44 57; fax: þ48 42 635 44 73. E-mail addresses: [email protected], [email protected] (A. Krokosz). 0969-806X/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radphyschem.2013.11.009
donor or acceptor capabilities of fullerenes. Because of their specific structure, fullerenes can be effective radical scavengers or prooxidants (Grebowski and Krokosz, 2010; Ratnikova et al., 2011; Kong and Zepp, 2012). Chemical modification of the fullerene carbon cage by an attachment of hydroxyl groups can be an effective way to overcome the low solubility of fullerenes in aqueous solutions. Due to the presence of hydroxyl groups on the surface of fullerenol molecules, it is possible for them to take part in many interactions, e.g. creating hydrogen bonds with biomolecules (Grebowski et al., 2013a, 2013b; Bhattacharya et al., 2012). Hydrogen bonds play an important role due to their frequent occurrence in biological systems and their role in biochemistry (Schuster and Wolschann, 1999; Krych and Gebicka, 2013; Moosavi-Movahedi et al., 2013). Ionizing radiation is known to cause irreversible alterations of protein conformation and function (Riley, 1994; Du and Gebicki, 2004;
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Rodacka et al., 2012). Protein damage due to indirect effects of irradiation could be minimized by optimization of the irradiation conditions e.g. by the use of antioxidants. Therefore the search for new, efficient radioprotectors is needed (Saloua et al., 2011). Due to their hydrophilic properties and the ability to scavenge free radicals, fullerenols may provide a serious alternative to the currently used pharmacological methods in chemotherapy, treatment of neurodegenerative diseases, and radiobiology. Additionally, due to the hollow spherical shape, fullerenols may be used as drug carriers (Grebowski et al., 2013c). The results obtained in many laboratories show that polyhydroxylated fullerenes, aka fullerenols or fullerols C60(OH)n, with more than 20 hydroxyl groups attached to carbon cage are watersoluble and exert mainly antioxidant/cytoprotective activity in biological systems (Cai et al., 2009; Vávrová et al., 2012) and may protect biomolecules and cells against ionizing radiation (Grebowski and Krokosz, 2010; Zhao et al., 2005; Bogdanović et al., 2008). However, there are a few studies regarding the toxic effects of fullerenol (Su et al., 2010; Wielgus et al., 2010). As irradiation of enzymatic proteins leads to their inactivation, we investigated the potential protective effect of fullerenol C60(OH) 30 against X-radiation on alcohol dehydrogenase and the effect of post-irradiation incubation in the presence or absence of fullerenol under aerobic conditions.
2. Experimental Fullerene C60 (99.5%) was purchased from SES Research (Houston, TX, USA). Alcohol dehydrogenase (ADH) (alcohol:NADþoxidoreductase, EC 1.1.1.1), molecular weight of 147 kDa from baker's yeast was purchased from Sigma-Aldrich (Poznan, Poland). All other chemicals were of analytical grade and were purchased from SigmaAldrich (Poznan, Poland) or POCH (Gliwice, Poland). All solutions were made with water purified by the Milli-Q system. 2.1. Synthesis of fullerenol C60(OH) 30 Polyhydroxylated fullerene derivative C60(OH) 30 was obtained by a method of Wang et al. (2005), with some modifications. Fullerene C60 and solid NaOH, in the presence of 30% H2O2 were ground in a glass mortar at a room temperature for 25 min until the color of the mixture turned to yellow-brown. Then, the incubation in a waterbath at 60 1C was carried out for 20 min. Deionized water was added to the reaction mixture and it was stirred for 24 h to dissolve the sludge. The solution of fullerenol was separated from any insoluble blend by centrifugation at 7000 g for 15 min (20 1C). After precipitation of fullerenol by methanol and resolubilization in deionised water, traces of NaOH were removed by ion-exchange chromatography with Amberlit MB-20. The structure of the obtained hydroxyl derivative of fullerene C60 was confirmed by IR spectrophotometry (NEXUS FT-IR spectrometer), 1H-NMR (Varian Gemini 200 MHz), 13C-NMR (Bruker Avance III 600 MHz) and mass spectroscopy MS-ESI (Varian 500 MS).
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enzyme was measured after 1 h from the end of irradiation or after 8 and 24 h of incubation at 25 1C in the dark. 2.3. The determination of enzyme activity Concentration of the ADH was determined on the basis of the absorbance measurements at 280 nm using the absorption coefficient of E1% 280 ¼ 14:6. ADH activity was measured by the method of Bonnichsen and Brink (1955). The activitiy of the enzyme was determined on the basis of the rate of reduction of NAD þ to NADH. The formation of NADH was estimated by the measurement of absorbance at λ ¼ 340 nm. Spectrophotometric measurements were carried out at room temperature in a CARY-1 apparatus (Varian, Melbourne, Australia). The resulting data were fitted by the linear or the linear-quadratic (LQ) model using Excel 2010 software (Microsoft Sp. z o.o., Warsaw, Poland). The doses corresponding to 37% of the enzyme activity (D37) and α, β values calculated from the fitted models were analyzed to compare the effect of fullerenol C60(OH) 30. Three independent experiments were performed for the each sample. 2.4. Calculations The radiation yield of Ginact for enzymes irradiated was calculated on the basis of the value of the dose D37 according to the formula cADH mol Ginact ¼ ρD37 J where cADH is the enzyme concentration in mol dm 3, D37 the irradiation dose (in Gy) at which the enzyme activity decreased to 37% of the initial activity and ρ the density of solution about 1 kg dm 3 (Rodacka et al., 2010). The radiation yield of dOH radicals reacting with ADH in the presence of fullerenol C60(OH) 30 was determined as described previously (Kowalczyk et al., 2008). Briefly, the radiation yield of d OH radicals reacting with enzyme in the presence of fullerenol was determined using the equation: G¼
kðOH þ EÞ ½E G0 ¼ ω G0 kðOH þ EÞ ½E þ kðOH þ FullOHÞ ½FullOH
where G0 – the yield of dOH radicals in the absence of fullerenol; in the atmosphere of air it was accepted that G0 ¼ 2.82 10-7 mol J-1; [FullOH] – the concentration of fullerenol; k – the appropriate rate constants of reaction of dOH radicals with enzyme [E] or fullerenol [FullOH]; for fullerenol - rate constant was 2.0 109 dm3 mol-1 s-1 (unpublished results); for ADH – rate constant was 1.8 1011 dm3 mol-1 s-1 (Buxton et al., 1988). ω – the fraction of dOH radicals reacting with enzyme in the competition with FullOH. If only the reaction of dOH radicals with enzyme was responsible for the expected inactivation, the yield would be calculated on the basis of the following equation: Gexp: inactiv: ¼
2.2. Conditions of irradiation
G 0 G ¼ ωG0inactiv: G0 inactiv:
where The ADH was dissolved in 0.02 M potassium phosphate buffer, pH 7.4, at the concentration of 0.1 mgprotein/mL (6.8 10-7 mol dm-3) and irradiated in the presence of or without fullerenol (FullOH) (75 mg/L). Solutions were irradiated in air using Stabilipan 2 an X-ray machine (Siemens, Germany) 195 kV, 18 mA, with an aluminum filter (2 mm). The dose rate estimated with a Fricke dosimeter was 4.1 Gy/min. Irradiation of samples was performed in a glass beaker with gentle mixing with magnetic bar. The activity of the
G0inactiv: – the yield of inactivation of the enzyme irradiated in the absence of fullerenol; Gexp: inactiv: – the expected yield of inactivation of the enzyme irradiated in the presence of fullerenol. It was assumed that molecular mass of fullerenol C60(OH) 30 was 1230 D and molar concentration 6.1 10-5 mol dm-3.
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2.5. Statistical analysis
log(%ActADH)= -(5.74x10-3D+3.86x10-5D2)+2
2.00
log (% of ADH activity)
The experimental values are expressed as the mean and the error bars indicate the standard error of the mean (SEM) calculated for each data point. All experiments were run 3–7 times. Significant differences between data were assessed by the paired Student's t-test with p o0.05. 3. Results and discussion
R2 = 0.9999 1.50
R2 = 0.9994 0.50 ADH
0.00
2.00
log(%ActADH) =-7.54x10-3D+2
log (% of ADH activity)
R2 = 0.9988 1.50
log(%ActADH) =-9.48x10-3D+2 R2 = 0.9966
0.50 ADH
0.00
0
ADH+fullerenol
20
40
60
80
100
Dose [Gy] Fig. 1. The effect of X-irradiation on the enzyme (alcohol dehydrogenase) irradiated in the absence or presence of fullerenol (75 mg/L) under air as the dependence of the logarithm of relative ADH activity on the radiation dose. Measurements were done 1 h after the end of irradiation. Each point is the mean of 3–7 independent experiments 7 SEM.
0
ADH+fullerenol
20
40
60
80
100
Dose [Gy]
2.00
log (% of ADH activity)
Alcohol dehydrogenase from baker's yeasts contains 2 cysteine residues (Cys-43, Cys-153) and 1 histidine residue (His-66) complexed with a Zn atom in the active site. ADH is effectively inactivated by radiation-induced dOH radicals (Kowalczyk et al., 2008; Rodacka et al., 2010). A significant role for inactivation susceptibility is played by the closest neighborhood of the active site; among others, by the number of more reactive amino acid residues such as Cys, Trp, Phe, His and Met. At the distance of 10 Å from the active site of ADH, there are 12 sensitive residues including 4 sulfur residues (PDB, code 2HCY). The results of ADH inactivation by X-radiation in the presence or absence of fullerenol are shown in Fig. 1. The dependence of log (% activity) on the radiation dose is linear, as it was obtained in the previous work (Kowalczyk et al., 2008). The presence of fullerenol at 75 mg/L did not change the character of the dependence but decreased the slope of the line (α) from 9.48 to 7.54 Gy 1. The slope of the line (inactivation coefficient) characterize radiosensitivity of the enzyme. It indicated that fullerenol could protect ADH with the efficiency of 20.5%. The post-irradiation incubation of the enzyme enhanced the inactivation of ADH as a result of previous irradiation (Fig. 2) and the linear-quadratic model (LQ) was used to describe the inactivation of ADH after 8 h and 24 h from the irradiation. The parameters calculated from the semi-logarithmic dose-effect curves (Fig. 2) for the dependence of relative ADH activity on the radiation dose for enzyme irradiated with X-rays without or with fullerenol at the concentration of 75 mg/L and incubated at 25 1C for 8 and 24 h are gathered in Table 1. It is worth to underline, that the concentration of fullerenol used in our work was rather high (75 mg/L) so the observed effects were explicitly pronounced. Moreover, the fullerenol at 75 mg/L and the time of incubation had no influence on the activity of unirradiated ADH up to 24 h of incubation at 25 1C in the dark (data not shown).
1.00
log(%ActADH)=-(8.00x10-3D+4.75x10-5D2)+2
1.00
log(%ActADH) =-(5.83x10-3D+6.44x10-5D2)+2 R2 = 0.9993
1.50
1.00
log(%ActADH)=-(8.17x10-3D+8.20x10-5D2)+2 R2 = 0.9995
0.50 ADH
0.00
0
ADH+fullerenol
20
40
60
80
100
Dose [Gy] Fig. 2. The effect of incubation on the enzyme (alcohol dehydrogenase) irradiated in the absence or presence of fullerenol (75 mg/L) under air as the dependence of the logarithm of relative ADH activity on the radiation dose, (A) after 8 h incubation; (B) after 24 h incubation from the end of irradiation. Samples were incubated at 25 1C. Each point is the mean of 3–7 independent experiments 7 SEM.
Table 1 The parameters calculated from the dose-effect curves for the dependence of logarithm of relative ADH activity on the radiation dose for enzyme irradiated with X-radiation without or with fullerenol at the concentration of 75 μg/ml and incubated at 25 1C. Time of incubation (h)
8 24
α 103 (Gy 1)
β 105 (Gy 2)
ADH
ADH þfullerenol
ADH
ADH þfullerenol
8.00 8.17
5.74 5.83
4.75 8.20
3.86 6.44
The linear-quadratic equation is well suited to fit experimental survival curves (Brenner et al., 1998) but it describes the postirradiation inactivation of ADH, the enzyme of oxidoreductase class, in an excellent way as well. Based on the linear-quadratic model, we can assume that the limiting slope (α) describing the linear part of the dose-effect curve, describes direct damage to the active site of the enzyme leading to its inactivation. However, the coefficient (β) for the dose-squared term of the curve indicates the participation of indirect damage to the enzyme leading to the inactivation of ADH. The post-radiation incubation of ADH did not result in the change of the α coefficient. However, the β coefficient increased 1.7-fold with the time of incubation independently of the presence of fullerenol (Table 1). This observation indicated the increase of post-radiation damage resulting from the modification of the structure of the enzyme by molecular oxygen from the air. As it was shown by Rodacka et al. (2010), the main role in radiosensitivity of ADH is played by -SH groups. Oxygen could oxidize –SH groups to disulfides in neutral pH (Bagiyan et al., 2003). The initial modifications in ADH structure by radiation could additionally facilitate oxidation of –SH groups by molecular oxygen. From the obtained in Figs. 1 and 2 dependences D37 values were determined, which served as a basis for the calculation of the
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Table 2 Doses D37 (the dose after which 37% of initial activity remains) and the radiation yields (Ginact) of inactivation of ADH irradiated with X-radiation without or with fullerenol at the concentration of 75 μg/ml and incubated at 25 1C; mean value7 SEM; significant difference compared to ADH (without fullerenol) is indicated by asterisk at po 0.05. Time of incubation (h)
D37 [Gy] ADH
Ginact [nmol/J] ADHþ fullerenol ADH
Without incubation 45.5 7 2.7 57.3 7 2.5n 8 43.0 7 6.3 54.9 76.7n 24 38.2 7 1.9 48.37 3.4n
ADHþ fullerenol
14.9 7 0.9 11.9 7 0.5n 15.8 7 2.4 12.4 7 1.6n 17.8 7 0.9 14.17 1.0n
radiation yield of inactivation according to the formula described in the Section 2.4. The radiation yield of inactivation of ADH irradiated with fullerenol was 20% lower than of ADH irradiated without fullerenol (Table 2). The efficiency of inactivation of ADH by radiation-generated d OH and O2 d radicals is 0.0350 and 0.0059, respectively (Rodacka et al., 2010) and we could take into attention mainly inactivation caused by dOH. In the present study, under conditions used during irradiation, 50% of dOH radicals could react with fullerenol and 50% could react with ADH as it was calculated according to the Section 2.4. Thus, it can be assumed that protective effect of fullerenol on the radiation inactivation of ADH was mostly due to scavenging d OH radicals by fullerenol. Guldi and Asmus, 1999, observed a negative correlation between the rate constant of scavenging dOH and the number of functional substituents in the range of different derivatives of fullerene C60. Hydroxyl radicals could abstract H atom or electron from the fullerenol as well as add to the double bonds. The more functional groups attached to the fullerene cage, the fewer number of π bonds in the molecule. This implies a decrease in possibility to scavenge free radicals and antioxidant capacity of fullerenol with increasing number of –OH groups attached to the carbon cage. However, our results revealed that highly hydroxylated fullerene exhibited an excellent solubility in water and kept their properties of scavenging dOH radicals simultaneously. The post-irradiation inactivation of the alcohol dehydrogenase irradiated in the absence or in the presence of fullerenol increased with the time of incubation (Table 2). However, fullerenol did not protect against post-irradiation damage as the Ginact for ADH irradiated with fullerenol was still 20% lower than for ADH irradiated without fullerenol after 24 h from irradiation. Fullerenol did not prevent the post-irradiation damage to the enzyme in our experimental system, which suggested the reduced ability of fullerenol to prevent the formation of protein peroxides. Protein peroxides form in high yields from proteins exposed to a wide range of ROS including hydroxyl radicals. Protein and peptide hydroperoxide groups have a half-life of several hours at room temperature and can inactivate enzymes. The first reaction product is a carbon-centered radical (Prd), rapidly converted in the presence of oxygen to a peroxyl radical (PrOOd) and finally to protein hydroperoxide (PrOOH). The precursor protein radicals can efficiently destroy antioxidants present in the experimental system (Du and Gebicki, 2004). Moreover, fullerenol could have associated with ADH by electrostatic, dipolar or Van der Waals interactions. The possibility of interaction of fullerenol with proteins was proved in our previous papers (Grebowski et al., 2013a, 2013b). Relative polar surface area of ADH is 0.375 so that one third of surface area of ADH which is 43577 Å (Rodacka et al., 2010) could interact by electrostatic and dipolar interactions with –OH groups of fullerenol. The interactions of proteins and nanoparticles (NPs) are reported in the newest papers considering protein corona
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(Fenoglio et al., 2011; Podila et al., 2012). These interactions influenced, among others, proteins activity, charge-transferinduced conformational changes and particles charge and reactivity. The ratio of the observed effectiveness of inactivation to the effectiveness of expected inactivation (t) was calculated according to the formula t¼
Ginactiv: obs: Gexp: inactiv:
The “t” parameter was 1.6 meaning that there was no or very little damage by secondary products of the reaction of the fullerenol with dOH radicals. We concluded that fullerenol C60(OH) 30 protected ADH against radiation inactivation due to simple competition for the d OH radicals and did not modify its activity by association with the protein as it was proved in our previous papers for erythrocyte membrane proteins (Grebowski et al., 2013a, 2013b).
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