Journal of Photochemistry and Photobiology B: Biology 148 (2015) 262–267
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Investigations of riboﬂavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity Chien-wei Cheng, Liang-yü Chen, Chan-wei Chou, Ji-yuan Liang ⇑ Department of Biotechnology, Ming-Chuan University, Gui-Shan 33343, Taiwan
a r t i c l e
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Article history: Received 27 January 2015 Accepted 30 April 2015 Available online 8 May 2015 Keywords: Superoxide dismutase LED Blue light NBT Riboﬂavin
a b s t r a c t Determination of the superoxide dismutase activity is an important issue in the ﬁelds of biochemistry and the medical sciences. In the riboﬂavin/nitro blue tetrazolium (B2/NBT) method, the light sources used for generating superoxide anion radicals from light-excited riboﬂavin are normally ﬂuorescent lamps. However, the conditions of B2/NBT experiments vary. This study investigated the effect of the light source on the light-excitation of riboﬂavin. The effectiveness of the photolysis was controlled by the wavelength of the light source. The spectra of ﬂuorescent lamps are composed of multiple colour lights, and the emission spectra of ﬂuorescent lamps made by different manufacturers may vary. Blue light was determined to be the most efﬁcient for the photochemical reaction of riboﬂavin in visible region. The quality of the blue light in ﬂuorescent lamps is critical to the photo-decomposition of riboﬂavin. A blue light is better than a ﬂuorescent lamp for the photo-decomposition of riboﬂavin. The performance of the B2/NBT method is thereby optimized. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Superoxide dismutase (SOD) is essential to many living organisms. This enzyme is one of the most important antioxidant defence mechanisms in microorganisms and plant cells that are exposed to oxygen . The antioxidant capacity of natural ingredients is also a signiﬁcant issue for foods with special dietary purposes . SOD, as a free radical scavenger, can convert superoxide anion radicals (O 2 ) into H2O2 and O2 in living cells. By scavenging O 2 , the oxidation of lipid membranes can be prevented . Superoxide anion radicals are intermediate products generated during oxidation or reduction. Formed from hydroxyl radicals or hydroxyl peroxide compounds, O 2 causes damage, inﬂammation, atherosclerosis and aging of cells [4,5]. Hence, the determination of SOD activities, either in vivo or in vitro, is an important topic in the ﬁelds of biochemistry and the medical sciences. Many methods, both direct and indirect, have been developed for the determination of SOD activity. However, direct assays for SOD determination are scarce because of their need for special apparatus, such as an electron paramagnetic resonance spectrometer (EPR). Indirect assays relying on the ability of SOD to inhibit O 2 -driven reactions are more widely applied in biochemical ⇑ Corresponding author. Tel.: +886 3 3507001x3772; fax: +886 3 3593878. E-mail address: [email protected] (J.-y. Liang). http://dx.doi.org/10.1016/j.jphotobiol.2015.04.028 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
laboratories . Beyer and Fridovich  investigated the effects of experimental variables on two indirect assays, the xanthine oxidase and cytochrome C method and the riboﬂavin/nitro blue tetrazolium method (B2/NBT). The B2/NBT method is considered simpler and is preferred for the quantiﬁcation of SOD activity in crude extracts . Riboﬂavin, also known as vitamin B2, is very sensitive to light. It decomposes after being irradiated by ultraviolet (UV) or visible light (420–560 nm) for a very short time , generating free radicals of reactive oxygen species (ROS), such as O 2 and singlet oxygen . The riboﬂavin photochemical treatment with blue light can be employed to inactivate E. coli with generated ROS [10,11]. Superoxide anion radicals generated from light-excited riboﬂavin can be utilized to examine the effect of luminance on light reactions of nitro blue tetrazolium (NBT) . In this study, NBT is used as an indicating scavenger to be reduced by O 2 . NBT reduction causes an increase in the absorbance at 560 nm in a process that may be inhibited by SOD. In addition, the B2/NBT method can be employed to evaluate the contents of phenolic compounds in functional foods through ROS scavenging. In the B2/NBT method, the light sources used for generating O 2 from light-excited riboﬂavin are normally ﬂuorescent lamps. A ﬂuorescent lamp is excited to generate ultraviolet rays by a low-pressure mercury vapour and argon. The inner wall of a glass tube is coated with a ﬂuorescent substance and stimulated by ultraviolet rays to generate a hybridized visible light. However,
C.-w. Cheng et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 262–267
the conditions and apparatuses for the B2/NBT experiments varied in the studies reviewed. The ﬂuorescent lamps used were of different speciﬁcations, including 13 W , 15 W [12,13], 20 W [7,14], 25 W , 30 W  and 40 W , and were illuminated over different distances and for different durations. The reactions were initiated by adding riboﬂavin at 2000 , 3000 , 4000  and 5000 lux  for the luminance of ﬂuorescent lamps. The effects of light source properties, such as colour and wavelength, on the light-excitation of riboﬂavin have been investigated using the B2/NBT assay . The ﬂuorescent lamp is excited to generate a hybridized visible light comprised of multiple colour lights. Thus, the properties of the light source could affect the riboﬂavin photochemistry, leading to incorrect conclusions from the B2/NBT assay. To ensure high accuracy, the widely accepted B2/NBT assay for the quantiﬁcation of SOD activity has to be validated. The current study developed an effective SOD assay from the B2/NBT method by applying a well-deﬁned light source to riboﬂavin photochemical reactions. The goal was to investigate the effects of light quality on the light-excitation of riboﬂavin as assayed by the B2/NBT method. The results thus obtained would promote the consistency of enzymatic measurements using photolysis reactions. 2. Materials and methods 2.1. Setup of illumination units The photo-induced reactions were performed in a plastic box (104 cm 74 cm 55 cm) with a light source. The box was made of white cardboard, and its outer surface was covered with black cloth. Three light-emitting diode (LED) tube lights (580 mm length) in red, green and blue (VITALUX T8HO LED tube lights, Vita LED Technologies Co., Tainan, Taiwan) and two ﬂuorescent lamps, Fluor-A (38 W, FHF38WEX, Taiwan Fluorescent Lamp Co., Taipei, Taiwan) and Fluor-B (30 W, FCL30D/28, China Electric MGF. Co., Taipei, Taiwan), were used as light sources. Irradiance was measured by the power of the electromagnetic radiation per unit area (mW/cm2) with radiometry and validated by a solar power meter (TM-207, Tenmars Electronics Co., Taipei, Taiwan). Luminance, a term used in photometry, was measured in lux (lx) or lm/m2 by a digital light meter (YF-170, Tenmars Electronics Co., Taipei, Taiwan).
Fig. 1. Visible spectra of ﬂuorescent and LED lamps used in this study.
The riboﬂavin (2.4 lM in 50 mM, pH 7.8 phosphate buffer) was irradiated by the ﬂuorescent lamps and LED tube lights at 1.0 mW/cm2 for 20 min. The absorbance of the illuminated riboﬂavin was detected at 200–800 nm by a UV/vis spectrometer (Lambda35, Perkin-Elmer). 2.4. Effects of light sources on the generation of O 2 with the B2/NBT method The reduction in NBT was determined using the method developed by Beauchamp and Fridovich . All solutions were 50 mM in phosphate buffer (pH 7.8). 3 mL of reactant was used, and the concentrations of riboﬂavin, methionine and NBT were 2.4 106 M, 0.01 M and 1.6 104 M, respectively. The distance between the reactant and the lamps was ﬁxed, and the irradiance was controlled. The reactant was illuminated by blue, green, yellow or red LED irradiation at 1.0 mW/cm2, by the ﬂuorescent lamps at 1.0 mW/cm2, and by the blue light irradiation at 0.1 mW/cm2 for 10, 20 or 30 min. For the control treatment, the reactant was kept in the dark. The photo-chemically reduction of riboﬂavin generated O 2 , which reduced NBT to form blue formazan, which can be detected at 560 nm (Lambda35, Perkin-Elmer). 2.5. Effects of light source on O 2 scavenging activity using gallic acid
2.2. Chemicals Gallic acid, l-methionine, monopotassium phosphate, potassium dihydrogen phosphate, riboﬂavin and SOD (S9697-15KU) were purchased from Sigma–Aldrich (St. Louis, MO). The SOD was assayed by Sigma–Aldrich using the xanthine oxidase/cytochrome C method . Nitro blue tetrazolium (NBT) was purchased from Bio Basic, Inc. (Markham, Ontario, Canada). Ultra-pure deionized water from a Milli-Q system was used as a solvent in this study.
Gallic acid was employed to determine the effects of the light source on the O 2 scavenging activity using the B2/NBT method described in Section 2.4. In brief, gallic acid (50 lL) was added to 3 mL reactant to ﬁnal concentrations of 0, 10, 20, 40, 60, 80 and 100 lg/mL. Then, the mixed solutions were subjected to Fluor-A or Fluor-B irradiation at 1.0 mW/cm2 or blue-light irradiation at 0.1 mW/cm2 for 20 min. Gallic acid can inhibit NBT reduction, and the scavenging capacity of the O 2 generated was calculated using the following equation, where A denotes the absorbance of the blue formazan measured at 560 nm.
2.3. Spectrometry of light sources and riboﬂavin
O 2 scavenging activityð%Þ ¼ ðAcontrol Asample Þ=Acontrol 100%
The emission spectra of the ﬂuorescent lamps and LED tube lights were measured using a UV–vis miniature ﬁbre optic spectrometer (USB4000 UV/Vis, Ocean Optics, USA) and were normalized, as shown in Fig. 1. The wavelengths of the emitted maxima of the blue, green, yellow and red lights were 463, 529, 589 and 632 nm, respectively, and the spectral widths at half height (W1/2) were 23, 31, 16 and 14 nm, respectively. The spectra of the ﬂuorescent lamps are usually comprised of several peaks, as shown in Fig. 1.
ð1Þ 2.6. Effects of blue light on SOD activity The effects of blue light on O 2 scavenging activity were examined with the B2/NBT method using SOD, as described in Section 2.4. In brief, (A) 50 lL SOD was added to 3 mL reactant, and the ﬁnal activity of SOD (1.0 unit/g) was used as a standard. Then, the mixed solutions were subjected to blue light irradiation
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at 0.08, 0.1 or 0.12 mW/cm2 for 10, 20 or 30 min. SOD activity was deﬁned as one unit of SOD having a 50% inhibition on the B2/NBT system. (B) 50 lL SOD was added to 3 mL reactant, and the ﬁnal activity of SOD was 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0 or 4.0 unit/g. The mixed solutions were exposed to blue light irradiation at 0.12 mW/cm2 for 10, 20 or 30 min. The optimal condition for the 50% inhibition of O 2 scavenging activity with SOD (1.0 unit/g) was measured using the B2/NBT method. 2.7. Statistics Data are represented by the mean ± standard deviation of three separate experiments. A homoscedastic two-sample t-test was employed to assess whether the two sets of measurements differed, and values of P < 0.05 were considered to be signiﬁcant. 3. Results 3.1. Effect of light quality on photo-decomposition of riboﬂavin The generation of O 2 from the intermediates during the decomposition of riboﬂavin in aqueous solution was detected using NBT reduction . As shown in Fig. 2, the absorbance of the NBT reduction increased with the generation of O 2 by the photochemical system in the presence of riboﬂavin. For the photochemical treatment, the decomposition of riboﬂavin increased with the irradiation time. The highest efﬁciency photochemical reaction of riboﬂavin was observed under blue light irradiation. The average photochemical effects of the green, yellow and red lights relative to the blue light were approximately 4.9%. 3.9% and 2.6%, respectively. These results indicate that the effectiveness of riboﬂavin photolysis is mainly determined by the wavelength of light used and that the light quality is associated with the photochemical reaction of riboﬂavin. 3.2. Spectra of riboﬂavin in photoreactions Fig. 3 shows the spectra (200–800 nm) of riboﬂavin measured by ﬂuorescence lamps and blue light irradiation. As observed, there were four absorption peaks, 224, 268, 373 and 445 nm, of riboﬂavin in the dark. The absorbance of riboﬂavin at 445 nm was dramatically decreased by blue light irradiation. The ﬂuorescent
Fig. 2. Effects of colour-light irradiation at 1.0 mW/cm2 on NBT reduction for 10, 20 and 30 min. Data are represented by mean ± standard deviation, where n = 3. Signiﬁcant differences (p < 0.05) between groups are indicated by different letters above the bar. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)
Fig. 3. Absorption spectra of riboﬂavin irradiated by different light sources at 1.0 mW/cm2 for 20 min.
lamps had a very small impact because the variations in the spectra at 445 nm were not signiﬁcant. Blue light irradiation for 20 min yielded the highest efﬁciency in the photo-decomposition of riboﬂavin. The spectra of riboﬂavin were measured during the course of colour illuminations in the photo-decomposition reactions . Irradiation with blue light showed the highest efﬁciency photo-decomposition of riboﬂavin, while the absorbance of riboﬂavin at 445 nm decreasing dramatically upon illumination. The green, yellow and red lights had negligible effects because the spectral changes were not signiﬁcant. Fig. 4 shows the effects of the irradiation of ﬂuorescent lamps and blue light on the NBT reduction during the photochemical reaction of riboﬂavin. As observed, the photochemical reaction of riboﬂavin increased with the irradiation time. The
Fig. 4. Effects of ﬂuorescent lamp irradiation at 1.0 mW/cm2 and blue light irradiation at 0.1 mW/cm2 on NBT reduction for 10, 20 and 30 min. Data are represented by mean ± standard deviation, where n = 3. Signiﬁcant differences (p < 0.05) between groups are indicated by different letters over the bar. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)
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photo-decomposition of riboﬂavin was lower under Fluor-A than under Fluor-B irradiation at 1.0 mW/cm2. 3.3. Effect of light source on
scavenging activity using gallic acid
Gallic acid is a tri-hydroxyl-benzoic acid. Phenolic compounds are considered the most important antioxidants in plants and plant-based foods . Many phenolic compounds, such as gallic acid, can remove free radicals through a process similar to SOD-catalysed reactions. The free radicals serve as a substrate and are scavenged by the phenolic compounds. Fig. 5 shows the variation in O 2 scavenging activity (%) using different levels of gallic acid. As seen in Fig. 5(A), the O 2 scavenging activity increased with the addition of gallic acid and was higher under Fluor-A irradiation than under Fluor-B. A characteristic concentration of chemicals, IC50, could be determined to provide 50% inhibition activity from the correlation curve of the scavenging activity to the concentration. The IC50 of gallic acid is deﬁned as the equivalent concentration of gallic acid that is able to remove 50% of the superoxide anion radicals. As shown in Fig. 5(B), the IC50 of gallic acid under Fluor-A and Fluor-B irradiation at 1.0 mW/cm2 was 25.7 lg/mL and 53.1 lg/mL, respectively, while the IC50 of gallic acid under blue-light irradiation at 0.1 mW/cm2 was 45.1 lg/mL. The IC50 of gallic acid under blue-light irradiation at 0.1 mW/cm2 and Fluor-B irradiation at 1.0 mW/cm2 were both insigniﬁcant.
Fig. 5. (A) O 2 scavenging activity of gallic acid under ﬂuorescent lamp irradiation at 1.0 mW/cm2 and blue light irradiation at 0.1 mW/cm2 for 20 min. (B) The IC50 of gallic acid under ﬂuorescent lamp irradiation at 1.0 mW/cm2 and blue light irradiation at 0.1 mW/cm2 for 20 min. Data are represented by mean ± standard deviation, where n = 3. Signiﬁcant differences (p < 0.05) between groups are indicated by different letters over the bar. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)
3.4. Effect of blue light on SOD detection The O 2 scavenging activity of one unit of SOD was determined by blue light irradiation using the B2/NBT method. As shown in Fig. 6(A), the O 2 scavenging activity of one unit of SOD decreased with an increase in the blue-light irradiation time for the photochemical treatment. The effect of the SOD activity on O 2 scavenging activity under blue-light irradiation were investigated using the B2/NBT method. As shown in Fig. 6(B), the O 2 scavenging activity increased with the SOD activity, and the 50% inhibition value of the O 2 scavenging activity at 0.12 mW/cm2 blue-light irradiation for 10, 20 and 30 min were 0.82, 0.90 and 0.97 unit SOD, respectively. The SOD activity is deﬁned as such that one unit of SOD that has a 50% inhibition on the B2/NBT system. The 50% inhibition of the O 2 scavenging activity under 0.12 mW/cm2 blue light irradiation for 30 min was 0.97 unit SOD, which is the optimal condition for the B2/NBT method in this study.
4. Discussion As shown in Fig. 2, riboﬂavin exhibits the highest efﬁciency photochemical degradation under blue light irradiation. The effects of green, yellow and red lights on riboﬂavin degradation were of low efﬁciency, indicating the absence of any charge transfer interaction between riboﬂavin and the phosphate buffered solution. Ahmad et al. used a mercury vapour ﬂuorescent lamp (emission at 405 and 435 nm) for riboﬂavin photolysis. A gradual decrease
Fig. 6. (A) Effect of one unit of SOD on O2 scavenging activity under 0.08, 0.1 and 0.12 mW/cm2 blue light irradiation for 10, 20 and 30 min. (B) Effect of SOD activity 2 on O 2 scavenging activity under 0.12 mW/cm blue light irradiation for 10, 20 and 30 min. Data are represented by mean ± standard deviation, where n = 3. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)
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in the absorbance of the aqueous phase at 445 nm indicated the loss of riboﬂavin. On the other hand, an increase in absorbance of a chloroform extract at 356 and 445 nm exhibited the formation of lumichrome and lumiﬂavin, respectively, with time . The effectiveness of the riboﬂavin photolysis is mainly determined by the wavelength of light. The photochemical degradation of riboﬂavin may proceed through the photoreduction of the isoalloxazine ring by electrons donated by the ribityl side chain . As shown in Fig. 3, the ratio between the absorptions at 445 nm and 373 nm indicates the efﬁciency of the photo-decomposition of riboﬂavin. The effectiveness values of dark, Fluor-A, Fluor-B and blue light irradiation at 1.0 mW/cm2 for 20 min were 1.65, 1.12, 1.07 and 0.79, respectively. The lower the effectiveness value, the higher the yield of the riboﬂavin photo-decomposition was. Blue light was found to be the most efﬁcient for the photo-decomposition of riboﬂavin, and the spectra of riboﬂavin were changed as a result of photo-degradation. Human eyes are very sensitive to visible light at the wavelength of 555 nm . The colours detected by human eyes depend on the speciﬁc wavelengths of the light sources. As shown in Table 1, Fluor-A and Fluor-B have same radiance intensity, but the illumination intensity of Fluor-A is higher than that of Fluor-B. At the same luminance, the IC50 of gallic acid under Fluor-A irradiation at 4000 lux (0.84 mW/cm2) treatment was 32.1 lg/mL, while that under Fluor-B irradiation at 4000 lux (1.0 mW/cm2) was 53.1 lg/mL. As shown in Fig. 5(B), the IC50 of gallic acid is 2.1-fold higher under Fluor-B than under Fluor-A irradiation at the same radiance intensity (1.0 mW/cm2). The scavenging capacity of O 2 by gallic acid is called SOD-like activity, and it can inhibit the riboﬂavin-mediated reduction of NBT. The IC50 of gallic acid is inversely proportional to the SOD-like activity. Hence, the SOD-like activity of gallic acid is 2.1-fold higher under Fluor-A than under Fluor-B irradiation at the same radiance intensity (1.0 mW/cm2). As seen in Fig. 4, the photochemical reaction of riboﬂavin was lower under Fluor-A than under Fluor-B irradiation at the same radiance intensity. The more O 2 were generated from the excited riboﬂavin, the more antioxidants were required for the elimination of O 2 in the B2/NBT method. The spectra of the ﬂuorescent lamps comprised multiple colour lights, as shown in Fig. 1. The emission spectra of ﬂuorescent lamps made by different manufacturers may vary. Different ﬂuorescent lamps might also generate different amounts of O 2 from the photo-decomposition of riboﬂavin at the same irradiance or luminance. To ensure high accuracy, the widely accepted B2/NBT assay for the quantiﬁcation of SOD activity by ﬂuorescent lamps has to be validated. As shown in Figs. 4 and 5(B), the photochemical reaction of riboﬂavin and the IC50 of gallic acid under Fluor-A irradiation at 1.0 mW/cm2 were similar to those under blue LED irradiation at 0.1 mW/cm2, as determined by the B2/NBT method in this study. After being light photo-energized, riboﬂavin is converted into excited triplet-state riboﬂavin. Superoxide anion or singlet oxygen is produced through the reaction of the excited triplet-state riboﬂavin with triplet oxygen [8,9]. However, the quality of blue light in the emission of the ﬂuorescent lamps plays a major role in the
Table 1 The measurements of luminance and irradiance of ﬂuorescence lamps and LED tube lights. Light source
Fluor-A Fluor-B Blue LED Green LED Red LED Irradiance (mW/cm2)
2070 1980 350 4560 750 0.5
4780 4000 670 10,190 1220 1.0
5940 5340 910 12,790 1830 1.5
8240 7160 1290 19,110 2460 2.0
photo-decomposition of riboﬂavin. The effectiveness of the riboﬂavin photolysis is controlled by the wavelength of the light source. The photo-decomposition of riboﬂavin under blue light at a low radiance intensity was selected to optimize the B2/NBT method. The emission spectra of coloured LEDs were always pure, clear and in a narrow wavelength range. The blue LED light showed the highest efﬁciency in the B2/NBT method. For the same energy dose (0.144 J/cm2), the effects of one unit of SOD on the O 2 scavenging activity after blue-light illumination for 30 min at 0.08 mW/cm2 and for 20 min at 0.12 mW/cm2 were 64.5% and 55.6%, respectively, as shown in Fig. 6(A). These results show that the light intensity (irradiance) has greater inﬂuence on the photo-decomposition of riboﬂavin than the irradiation time. 5. Conclusions The effectiveness of photolysis is controlled by the wavelength of the light source. The quality of the blue light in ﬂuorescent lamps is critical to the photo-decomposition of riboﬂavin. In this study, blue light was found to exhibit the highest efﬁciency photochemical reaction of riboﬂavin. It is concluded that the blue LED light is better than the ﬂuorescent lamp for the photo-decomposition of riboﬂavin. Irradiation by blue light at 0.12 mW/cm2 for 30 min is determined to be the optimal condition for the B2/NBT method. Acknowledgment The ﬁnancial support in this work is partially from the Ministry of Science and Technology, Taiwan, under Contracts No. MOST 103-2113-M-130-001 (Grant to L.-Y. Chen). References  L.-S. Lai, P.-C. Chang, C.-T. Chang, Isolation and characterization of superoxide dismutase from wheat seedlings, J. Agric. Food Chem. 56 (2008) 8121–8129.  L.Y. Chen, C.W. Cheng, J.Y. Liang, Effect of esteriﬁcation condensation on the Folin–Ciocalteu method for the quantitative measurement of total phenols, Food Chem. 170 (2015) 10–15.  R. Zimmermann, L. Flohe, U. Weser, H.J. Hartmann, Inhibition of lipid peroxidation in isolated inner membrane of rat liver mitochondria by superoxide dismutase, FEBS Lett. 29 (1973) 117–120.  B. Halliwell, J.M. Gutteridge, Role of free radicals and catalytic metal ions in human disease: an overview, Methods Enzymol. 186 (1990) 1–85.  J.W. Juen, H.L. Jian, J.Y. Liang, The effect of illuminance on light induced reduction of nitro blue tetrazolium, MC-Trans. Biotechnol. (2010).  J.K. Donnelly, K.M. McLellan, J.L. Walker, D.S. Robinson, Superoxide dismutases in foods. A review, Food Chem. 33 (1989) 243–270.  W.F. Beyer Jr., I. Fridovich, Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions, Anal. Biochem. 161 (1987) 559–566.  P.B. Ottaway, Stability of vitamins in food, in: The Technology of Vitamins in Food, Chapman and Hall, London, 1993, pp. 233–244.  Y. Lin, R.R. Eitenmiller, W.O. Landen, Riboﬂavin, in: Vitamin Analysis for the Health and Food Sciences, CRC Press, 2008, pp. 329–360.  J.-Y. Liang, C.-W. Cheng, C.-H. Yu, L.-Y. Chen, Investigations of blue lightinduced reactive oxygen species from ﬂavin mononucleotide on inactivation of E. coli, J. Photochem. Photobiol., B 143 (2015) 82–88.  J.-Y. Liang, J.-M.P. Yuann, C.-W. Cheng, H.-L. Jian, C.-C. Lin, L.-Y. Chen, Blue light induced free radicals from riboﬂavin on E. coli DNA damage, J. Photochem. Photobiol., B 119 (2013) 60–64.  C. Beauchamp, I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Anal. Biochem. 44 (1971) 276–287.  M. Behnamnia, K.M. Kalantar, J. Ziaie, The effects of brassinosteroid on the induction of biochemical changes in Lycopersicon esculentum under drought stress, Turk. J. Bot. 33 (2009) 417–428.  H.-S. Kim, C.-D. Jin, Polyamines as antioxidant protectors against paraquat damage in radish (Raphanus sativus L.) cotyledons, J. Plant Biol. 49 (2006) 237–246.  A. Boonmee, C. Srisomsap, A. Karnchanatat, P. Sangvanich, An antioxidant protein in Curcuma comosa Roxb. Rhizomes, Food Chem. 124 (2011) 476–480.  S. Sundaram, S. Anjum, P. Dwivedi, G. Rai, Antioxidant activity and protective effect of banana peel against oxidative hemolysis of human erythrocyte at different stages of ripening, Appl. Biochem. Biotechnol. 164 (2011) 1192–1206.
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Copper (Cu) is essential both for its role in antioxidant enzymes, like Cu/zinc (Zn) superoxide dismutase (SOD) and ceruloplasmin, as well as its role in lysyl oxidase, essential for the strength and integrity of the heart and blood vessels. With suc
Amino acid networks (AANs) analysis is a new way to reveal the relationship between protein structure and function. We constructed six different types of AANs based on iron superoxide dismutase (Fe-SOD) three-dimensional structure information. These