Accepted Manuscript

A new formula to calculate activity of superoxide dismutase in indirect assays

Chen Zhang, Marieke E. Bruins, Zhi-qiang Yang, Shu-tao Liu, Ping-fan Rao

PII: S0003-2697(16)00114-7
DOI: 10.1016/j.ab.2016.03.014
Reference: YABIO 12341

To appear in: Analytical Biochemistry

Received Date: 21 January 2016

Accepted Date: 18 March 2016

Please cite this article as: C. Zhang, M.E. Bruins, Z.-q. Yang, S.-t. Liu, P.-f. Rao, A new formula to
calculate activity of superoxide dismutase in indirect assays, Analytical Biochemistry (2016), doi:
10.1016/j.ab.2016.03.014.

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1 A new formula to calculate activity of superoxide dismutase in indirect assays

2 Chen Zhang1,2, Marieke E. Bruins2, Zhi-qiang Yang1, Shu-tao Liu1,*, Ping-fan Rao3

3 Institute of Biotechnology, Fuzhou University, Gong-Ye-Lu 523, Fuzhou, Fujian, China.1

4 Wageningen UR Food & Biobased Research, Bornse Weilanden 9, 6708WG Wageningen, the Netherlands.2
5 CAS.SIBS-Zhejiang Gongshang University Joint Center for Food & Nutrition Research, Hangzhou, 310035,
6 China.3

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7 Abstract: To calculate superoxide dismutase (SOD) activity rapidly and accurately by indirect SOD

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8 assays, a formula based on the ratio of the catalytic speed of SOD to the reaction speed of the

9 indicator with superoxide anion was deduced. The accuracy of this formula was compared with the

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10 conventional formula based on inhibition in five indirect SOD assays. The new formula was validated

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11 in almost the entire SOD activity range, whereas the conventional formula was validated only during
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12 inhibition of 40% to 60%. This formula might also be used for the assays of other enzymes.

13 Highlights:
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14 • A formula based on VSOD / VIndicator was induced for indirect SOD assays.
15 • New formula validated in the range of 0-85% inhibition.
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16 • New formula was validated by five indirect SOD assays that were universally used.
• New formula may be applied to other enzyme activity assays using indirect method.
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17

18 Keywords: SOD; hydroxylamine hydrochloride; epinephrine; nitroblue tetrazolium; cytochrome C;

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19 WST
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20
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*: Corresponding Author: Shutao Liu. Email: stliu@fzu.edu.cn

Abbreviation: SOD, Superoxide dismutase; NBT, Nitroblue terazolium;
1 WST, Water-soluble tetrazolium.
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21 1 Introduction

22 Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of superoxide anion (O2-•)

23 into oxygen and hydrogen peroxide, which was found in 1969 [1]. Due to its bioactivity in the

24 scavenge of superoxide radicals, SOD is considered as one of the most important antioxidant

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25 metalloenzymes [2]. The insufficiency of SOD in metabolism may result in excessive amount of

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26 reactive oxygen species (ROS), which can damage macromolecules (protein, lipids, and DNA) and

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27 cell membranes [3, 4].

28 SOD is so important in the studies of ROS and antioxidant that a rapid and accurate method to

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determine its activity is required. As the substrate, O2-• is unstable, SOD activity is mainly determined
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29

30 by indirect methods. Most of these indirect assays depend upon competition between SOD and O2-•
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31 scavenging indicator, such as hydroxylamine hydrochloride, nitroblue tetrazolium (NBT), cytochrome

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32 C or water-soluble tetrazolium (WST) [1, 5-8]. These methods are widely used as they are convenient
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33 and affordable. However, close to 50% inhibition is critical to obtain precise activity in these assays,

34 and it is very difficult to obtain 50% inhibition by several determinations for an unknown SOD
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35 sample because SOD activity is proven to be nonlinear to inhibition [9]. To linearize inhibition to
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36 SOD activities, modifications were made by using an inhibition to log (SOD activity) standard curve
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37 [10], a reciprocal of inhibition to SOD activity standard curve [11], but these improvement do not

38 fundamentally change the accuracy of these assays. In fact, instead of inhibition, SOD activity was

39 found to be linear to the ratio of SOD’s catalytic speed to the indicator’s reaction speed (VSOD/VIndicator)

40 in a cytochrome C assay, providing a new route to calculate SOD activity [12]. Nevertheless, this

41 discovery is not applied in current SOD assays although it was suggested 40 years ago.
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42 Based on the linearity of SOD activity to VSOD/VIndicator, we deduced a new formula. Furthermore, the

43 accuracy of this formula was compared with the conventional formula in five indirect SOD assays.

44 The data of hydroxylamine hydrochloride assay was collected practically, and data from other assays

45 were gathered from the literature [1, 5-8].

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46 2 Materials and Methods

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47 The SOD sample for hydroxylamine hydrochloride assay was prepared according to the previous

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48 procedure [13]. The stock solution of the SOD sample was 4000 U/mL; it was then diluted to

49 concentrations of 5, 10, 20, 50, 100 and 200 U/mL and used as standards.

50 3 Results
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51 3.1 Deduction of a new formula to calculate SOD activity
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52 In the indirect SOD assay, considering the dilution in the determination system, the conventional

53 formula for the calculation of SOD activity presented by absorbance is shown below:
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A0 − A1
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System Volume
54 SOD activity = ÷ 50% × × Dilution Factor F.1
A0 Sample Volume
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55 Here, A0 is the absorbance in the absence of SOD, A1 is the absorbance in the presence of SOD, and

56 the enzymatic activity that causes 50% inhibition in the system is defined as one unit. In other words,
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57 the SOD activity causing A1 = 0.5 A0 is defined as 1 U.

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58 As absorbance is linear to the concentration of the colored compound (the product of the reaction of

59 indicator and superoxide anion), A0 and A1 are related to the reaction rate of the indicator with total

60 O2-• (VTotal) and fractional O2-• reacting with the indicator (VIndicator). Because VSOD= VTotal-VIndicator, the

61 difference of absorbance (A0-A1) is with respect to the catalytic rate of SOD (VSOD). Therefore,
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A0 − A1
62 VSOD/VIndicator = A1
.

63 It was found by [12] that VSOD = KSOD [SOD] [O2-•] and VIndicator = KIndicator [Indicator] [O2-•]. Here,

64 [SOD], [Indicator], and [O2-•] represent the concentration of SOD, indicator and O2-• respectively.

K SOD [SOD] [O 2 •]
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65 Therefore, VSOD/VIndicator = K , in which [O2-•] can be eliminated. Combined with the

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[Indicator ] [O 2 •]
-
Indicator

A0 − A1 K Indicator [Indicator ] A0 − A1
66 above formula of VSOD/VIndicator = A1
, it can be deduced that [SOD] = K SOD A1
.

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K Indicator [Indicator ]
67 Because one unit of SOD activity is defined as when A1 = 0.5 A0, here the constant, K SOD
, is

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68 defined as 1U. Therefore, considering the dilution rate, the SOD activity of the original sample is

69 presented as the following new formula:

70 SOD activity =
A0 − A1 System Volume
×
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× Dilution Factor F.2
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A1 Sample Volume

A0 − A1
71 Different from ‘inhibition’, we consider as a correlation factor (CF). This correlation factor
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A1

72 presents the ratio of SOD catalytic ability to indicator reaction ability. The validities of F.1 and F.2 in
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73 various SOD assays were then compared by data from our own experiments and data obtained from
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74 the literature [1, 7, 8, 14].

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75 3.2 Accuracy of the two formulas in five indirect SOD assays

76 3.2.1 Hydroxylamine hydrochloride assay

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77 In this assay, the absorbance data were collected practically. SOD samples with activities of 5, 10, 20,
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78 50, 100, 200 U/mL were used as standards. Absorbance in the absence or the presence of the SOD

79 standards (A0 and A1) was measured. The SOD activity was plotted against inhibition or CF according

80 to F.1 and F.2, and the theoretical curves of the two formulas were presented to identify their accuracy.

81 Fig. 1a shows the results of the conventional formula (F.1) and new formula (F.2) fitted to the
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82 measured data. The value of F.1 largely deviates from the actual activity of SOD when inhibition is

83 higher than 50%, whereas that of F.2 starts to deviate only at inhibition above 84%. The linearity of

A0 − A1
84 enzyme activity to CF, calculated by A1
, is shown in Fig. 1b.

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85
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86 Fig.1 SOD activities determined by hydroxylamine hydrochloride assay. ▲: experimental data. (a) Comparison of
87 theoretical values of two formulas. —: F.1; ---: F.2；(b) Linearity of correlation factor to SOD activity. —: Trend line.
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88 3.2.2 Epinephrine assay

89 McCord and Fridovich [1] were the first to publish the SOD assay. In their article, epinephrine was
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90 proven to be competent as an indicator for SOD assay. Based on Fig. 8 of their article [1], the

91 absorbance difference ∆A at t=2 min can be calculated for the blank, 11 ng SOD and 45 ng SOD. The
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92 values are: ∆A0=0.0106, ∆A1=0.0050, and ∆A2=0.0019, respectively. According to F.1, the activities

93 of 11 ng SOD and 45 ng SOD are 1.06U and 1.64U, respectively. However, according to F.2, values
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94 of 1.12U and 4.58U are obtained, respectively. When looking at the ratio of the SOD amount, which is

95 11 ng: 45 ng, the latter formula shows more reasonable results. This again illustrates the better

96 reliability of F.2 at higher inhibition rates.

97 3.2.3 NBT assay

98 Experimental data of NBT assay, cited from Fig. 6 of Kono [7], is used to demonstrate the accuracy of
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99 the two formulas by coinciding the zero point and 50% inhibition. Similar as Fig.1 (See Appendix

100 Fig.1), all experimental data are close to the curve of F.2, whereas only the point close to the 50%

101 inhibition follows the line of F.1.

102 3.2.4 Cytochrome C assay

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103 Experimental data of cytochrome C assay, cited from [14], was also applied for testing the accuracy of

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104 these two formulas. Similar as Fig.1 (See Appendix Fig.2), the same conclusion can be made as that

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105 from the hydroxylamine hydrochloride assay and the NBT assay. F.2 is a better fit to the experimental

106 data.

107 3.2.5 WST assay

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108 Experimental data from the WST assay reported by Peskin and Winterbourn [8] was also applied for
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109 validation. Similar as Fig.1 (See Appendix Fig.3), F.2 is a better fit to the experimental data and the

110 enzyme concentration is linear to CF that R2 is close to 1.

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111 4 Discussion

112 Based on the above figures, inhibition is proven to be nonlinear to SOD activity or concentration, and
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A0 − A1
113 thus the calculation results are only accurate when they are close to 50% inhibition. In F.1, as A0
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114 is linear to VSOD , and therefore equals to KSOD [SOD] [O-2•], in which dynamic [O-2•] will result in
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A0 − A1
115 inaccuracy in the assay system. In the new formula (F.2), A0 is linear to VSOD/VIndicator, which

K SOD [SOD] [O 2 •]
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116 equals to K [Indicator ] [O 2 •]

- . Here, the influence of [O-2•] was eliminated that the new formula fits the
Indicator

117 mechanism of indirect SOD assay and is superior to the currently used formula. The constant,

K Indicator [Indicator ]
118 K SOD
, can be used to covert activities of SOD samples that were determined by different

119 indirect assays. When CF is in the range of 0~4 (inhibition 0~80%), the calculated result can represent
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120 the actual value precisely. At higher inhibition values, the calculated values start to deviate from the

121 measured ones. Deviations in this new formula occur only above 80% inhibition. The deviation is

122 probably due to an increase in the amount of catalytic product, hydrogen peroxide , which inhibits

123 SOD activity [9, 15].

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124 This formula may also be applied to other enzyme activity assays with similar mechanisms, such

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125 as the determination of the activity of the competitive inhibitors of angiotensin converting enzyme

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126 [16].

127 5 Conclusion

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Based on the ratio of SOD catalytic speed to indicator reaction speed instead of inhibition rate,
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128

129 the accuracy of the new formula is tenable in five indirect SOD assays that were commonly used.
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130 SOD activity obtained from different indirect SOD assays is easily comparable, which facilitates the
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131 comparisons among the SOD studies and the market value of SOD. This formula may also be applied
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132 to other enzyme activity assays with similar mechanisms.

133 6 Acknowledgements:
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134 This study was supported by Natural Science Foundation of China (No.31071497，31271859),
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135 National Basic Research Program of Science and Technology Ministry of China (No.2010CB530605).
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136 7 References
137 [1] J.M. McCords, I. Fridovich, superoxide dismutase, The journal of Biological Chemistry 244 (1969) 6049-6055.
138 [2] F. Zeinali, A. Homaei, E. Kamrani, Sources of marine superoxide dismutases: Characteristics and applications,
139 International Journal of Biological Macromolecules, 79 (2015) 627-637.
140 [3] R. Dixit, H. Mukhtar, D.R. Bickers, Studies on the Role of Reactive Oxygen Species in Mediating Lipid Peroxide
141 Formation in Epidermal Microsomes of Rat Skin, Journal of Investigative Dermatology, 81 (1983) 369-375.
142 [4] M. Tarr, F. Samson, D. Borg, Oxygen Free Radicals and Tissue Injury, Oxygen Free Radicals in Tissue Damage,
143 Birkhäuser Boston1993, pp. 12-53.
144 [5] E.F. Elstner, A. Heupel, Inhibition of nitrite formation from hydroxylammoniumchloride: A simple assay for superoxide
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145 dismutase, Analytical Biochemistry, 70 (1976) 616-620.
146 [6] L. Flohé, F. ötting, Superoxide dismutase assays, in: P. Lester (Ed.) Methods in Enzymology, Academic Press1984, pp.
147 93-104.
148 [7] K. Yasuhisa, Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide
149 dismutase, Archives of Biochemistry and Biophysics, 186 (1978) 189-195.
150 [8] A.V. Peskin, C.C. Winterbourn, A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt
151 (WST-1), Clinica Chimica Acta, 293 (2000) 157-166.
152 [9] S. Goldstein, C. Michel, W. Bors, M. Saran, G. Czapski, A critical reevaluation of some assay methods for superoxide

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153 dismutase activity, Free Radical Biology and Medicine, 4 (1988) 295-303.
154 [10] I. Durak, Z. Yurtarslanl, O. Canbolat, Ö. Akyol, A methodological approach to superoxide dismutase (SOD) activity
155 assay based on inhibition of nitroblue tetrazolium (NBT) reduction, Clinica Chimica Acta, 214 (1993) 103-104.

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156 [11] L.E. Ilouno, E.N. Shu, G.E. Igbokwe, An improved technique for the assay of red blood cell superoxide dismutase
157 (SOD) activity, Clinica Chimica Acta, 247 (1996) 1-6.
158

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[12] Y. Sawada, I. Yamazaki, One-electron transfer reactions in biochemical systems. VIII. Kinetic study of superoxide
159 dismutase, BBA - Enzymology, 327 (1973) 257-265.
160 [13] J. Guo, Y. Chen, B. Yuan, S. Liu, P. Rao, Effects of Intracellular Superoxide Removal at Acupoints with TAT-SOD on
161 Obesity, Free Radical Biology and Medicine, 51 (2011) 2185-2189.

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162 [14] Beyer J.R., W. F., I. Fridovich, Assaying for superoxide dismutase activity: Some large consequences of minor changes
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163 in conditions, Analytical Biochemistry, 161 (1987) 559-566.
164 [15] G.D. Mao, P.D. Thomas, G.D. Lopaschuk, M.J. Poznansky, Superoxide dismutase (SOD)-catalase conjugates. Role of
165 hydrogen peroxide and the Fenton reaction in SOD toxicity, J. Biol. Chem., 268 (1993) 416-420.
166
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[16] D.W. Cushman, M.A. Ondetti, Design of angiotensin converting enzyme inhibitors, Nature America Inc., 5 (1999)
167 1110-1112.
168
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