Anal Bioanal Chem (2011) 401:381–386 DOI 10.1007/s00216-011-5070-8
TECHNICAL NOTE
Determination of superoxide dismutase and SOD-mimetic activities by a chemical system: Co2/H2O2/lucigenin T. V. Zhidkova & E. V. Proskurnina & E. A. Parfenov & Yu. A. Vladimirov
Received: 10 January 2011 / Revised: 22 April 2011 / Accepted: 27 April 2011 / Published online: 15 May 2011 # Springer-Verlag 2011
Abstract The bright chemiluminescence has been observed in the system: Co2+/hydrogen peroxide/lucigenin. The chemiluminescence intensity was directly proportional to either cobalt, hydrogen peroxide, or lucigenin concentrations. A procedure of determination of superoxide dismutase (SOD) activity by the chemiluminescence method in the cobalt–hydrogen peroxide–lucigenin system at pH 8.5 is suggested. A linear dependence was established between a relative chemiluminescence intensity and SOD concentration in the range of SOD concentrations between 0 and 4.5 nM, c1/2 =0.8 nM. The determination of SOD activity was performed in several tissue samples (rat plasma, erythrocyte hemolysate, and liver mitochondria). A technique of tissue sample preparation with the use of thermal inactivation of interfering proteins at 60 °C was used. The method was successfully applied for comparison of the efficiency of SOD mimetics. Keywords Superoxide dismutase/*analysis . Chemiluminescent measurements . Lucigenin . SOD mimetics . Animals
Introduction Superoxide dismutase (SOD), an enzyme of the protective system of an organism, neutralizes superoxide radicals by T. V. Zhidkova (*) : E. V. Proskurnina : Y. A. Vladimirov Faculty of Fundamental Medicine, Lomonosov Moscow State University, Moscow 117192, Russia e-mail:
[email protected] E. A. Parfenov N.N. Blokhin Russian Cancer Research Center, Moscow 115478, Russia
means of their dismutation to hydrogen peroxide and triplet oxygen, thus maintaining the superoxide radical concentration at a stationary and safe-for-cell level. Superoxide radical (•OO−) spontaneously dismutates rather quickly to oxygen, O2, and hydrogen peroxide, H2O2. On the other hand, superoxide reacts still more quickly with some other molecules, such as nitrogen oxide NO, to form peroxynitrite, thus initiating lipid peroxidation. For clinical purposes and for study of pathologies and treatment of specific diseases, the problem of SOD activity determination often arises. To determine SOD activity, different methods have been used. The following systems are most often exploited for superoxide radical generation: & & & & &
Xanthine and xanthine oxidase [1–3] NADH and phenazine methosulfate [4, 5] Riboflavin photooxidation [6] Adrenalin autooxidation [7, 8] Luminol photooxidation [9]
The following methods were applied for control of the amount of superoxide produced in the presence of SOD: & & &
Photometry [1, 4, 10, 11] Chemiluminescence (CL) [3, 9, 12] Electrochemical detection [13, 14]
Recent progress in research area of SOD activity determination is focused to the development of microtiter [12, 15] and chip-based systems [16]. The methods with the use of xanthine oxidase and NADH demand expensive reagents for superoxide radical generation. Moreover, in the majority of the described methods, the activity of SOD is determined at high pH (9–11), while the analysis of biological objects and drugs should be preferably performed at pH, close to physiological, in order to have an opportunity to estimate what really occurs in living cells and biological fluids. In the
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present paper, we propose to use the system H2O2 +Co2+ as a source of superoxide radicals and lucigenin-dependent chemiluminescence as a method to monitor the concentration of superoxide radicals. The SOD activity in this system was determined by the degree of the luminescence quenching upon the addition of the analyte. The SOD activity of several new SOD mimetics was investigated by this method, as well as of some biological objects.
Materials and methods Chemicals The following chemicals were used: KH2PO4 (≥98.0%, Sigma); lucigenin (Sigma-Aldrich); hydrogen peroxide (30%, Aldrich); CoCl2·6H2O (98.0–102.0%, Sigma); SOD (4,470 units/mg, Sigma); SOD mimetics SOD copper model C18H16N6O4Cu∙1,5H2O; and complex compounds N-acetyl- L -cysteine with a Co (II) ion C 5 H 7 NO 3SCo∙5,5H2 O, N-acetyl- L -cysteine with a Co (III) ion C10H15N2O6S2Co∙3H2O, and N-acetyl-L-cysteine with a Zn (II) ion C10H18N2O10S3Zn∙8H2O (RF Patent No. 2265608 of July 26, 2004). Equipment Chemiluminescence was measured on a SmartLum-5773 chemiluminometer (Inter-Optika-C, Russia) at room temperature (20 °C). To interface the chemiluminometer with a computer, an original version 3.3 PowerGraph software product designed by D.Y. Izmaylov was used. A 4-mL cuvette of polysterene was used to measure the CL emission.
T.V. Zhidkova et al.
Detection limits, cmin, were calculated based on the standard deviation (SD) of the response r and the slope of the regression equation or sensitivity S. cmin was calculated by the equation 3r/S. The precision of the method was verified in terms of intra-precision and inter-precision using a solution of 10 μM SOD copper model. The results were expressed as the covariation (CV%=(SD/mean)·100) of the c1/2. To estimate the repeatability, three samples of SOD copper model were dissolved in 1 ml of distilled water. Each of the solutions was diluted at 10 μM SOD copper model. For each of the solutions, c1/2 was measured. The intermediary precision was calculated from a 10-μM probe solution on three different days. On each day, a new solution was prepared and analyzed. The data were expressed as RSD. Determination of SOD activity in rat plasma, erythrocytes, and liver mitochondria The experiments were carried out on 2–3-month-old male outbred albino rats, n=3 (302±18 g). The rats were decapitated under ether. A heparin-stabilized (2 units/ml) blood sample was centrifuged at 900×g, plasma was isolated, and erythrocytes were washed twice with 0.9% NaCl and diluted with distilled water (1:10). Rat liver mitochondria were isolated using the differential centrifugation method. The protein concentration was determined by the biuretic method. The obtained samples were stored at −20 °C. To remove the components preventing SOD activity determination (catalase, ascorbic acid, and glutathione), the specimens were kept for 10 min at 60 °C (the activity of SOD under thermal treatment reduces only at above 75 °C [17]). The activity of SOD was determined as described in the previous method.
Study of the effect of SOD and SOD mimetics Thermal inactivation of SOD The reagents are listed in the order they were added into the cuvette. Eight hundred seventy-five microliters (volume of SOD) of 100 mM phosphate buffer solution (PB) pH 8.50± 0.02, 15 μl of 20 mM CoCl2, 100 μl of 1 mM lucigenin, from 0 to 18 μl of 250 nM SOD, and 10 μl of 0.30 M hydrogen peroxide were added to a CL cuvette. Concentrations of final solutions were as follows: 0.30 mM CoCl2, 0.10 mM lucigenin, 0 to 4.50 nM SOD, and 3.00 mM hydrogen peroxide. The obtained solution was placed into the chemiluminometer, and CL was recorded for 10 min. Concentrations of SOD mimetics were varied similarly. Method validation Assay was validated considering linearity, valid range, detection limits, precision, and accuracy of the method.
For a thermal inactivation of SOD (a solution in distilled water and plasma, erythrocyte, and mitochondria samples), the solutions were kept for 10 min on water bath at 95 °C. Statistical analysis Data were expressed as the mean± SD of the mean of triplicates from three independent experiments. To compare mean values, we used modified Student’s test. To compare the variances, we used Fisher’s test. The data were linearized with the least-square procedure. The adequacy of the linear model was estimated by comparing the adequacy variance with blank experiments variance.
Determination of superoxide dismutase and SOD-mimetic activities
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The scheme of hydrogen peroxide decomposition in the presence of cobalt ions is as follows:
Results and discussion The chemiluminescence in the system: Co2+, H2O2, and lucigenin In the presence of cobalt, hydrogen peroxide decomposed to form superoxide radical (Fenton reaction), and lucigeninactivated chemiluminescence (Luc-CL) was used for superoxide radical detection [18, 19]. In that system, a luminescence was also observed in the presence of luminol as a CL activator [20, 21]. Superoxide radical can be also determined by the CL-reagent coelentrazine [22] and MCLA (6-(4-methoxyphenyl)-2-methylimidazo[1,2-a] pyrazin-3(7H)-one, luciferin analog) [23]. However, lucigenin is most suitable and more specific probe for superoxide radical [18]. Lucigenin shows extremely intense chemiluminescence at pH≥9, which is far greater than physiological conditions. Therefore, for the restriction of biological samples, the pH used was complex compound N-acetyl-L-cysteine with a Zn (II) ion>complex compound N-acetyl-L-cysteine with a Co (II) ion>complex compound N-acetyl-L-cysteine with a Co (III) ion. However, even the most effective mimetic, SOD copper model, was 30 times less effective than SOD. The obtained results enabled to calculate correctly the dose of reagents for further in vivo investigations. Validation Analytical parameters of SOD and SOD-mimetic activity determination are presented in Table 1. The experimental values obtained for the determination of SOD copper model are presented in Table 2. Difference between intra-assay and inter-assay variances is statistically insignificant (α=0.05). Therefore, the reproducibility is also the same. No statistically significant differences (α=0.05) between the values of c1/2 are found. However, if the variations in pH values are greater than 0.02, it leads to variations in CL emission. In such a case, the data are not
4 3
A0/A
Study of the effect of SOD and SOD mimetics
2 1
Table 2 Statistical results of precision of c1/2 of SOD copper model (n=3 for each day) c1/2 ±SD (nM) Intra-assay (n=3) Day 1 Day 2 Day 3 Inter-assay (n=3)
95±8 90±3 91±4 92±3
CV (%)
8 3 5 3
0
0
50
100
150
Cp (µg/ml)
Fig. 2 Intensity ratio of the maximum CL without and with sample as a function of the amount of rat plasma protein (diamonds), erythrocyte fraction (squares), and liver mitochondria fraction (triangles). One thousand microliters of a 100-mM PB solution, pH 8.5, contains 0.30 mM CoCl2, 0.10 mM lucigenin, 0.3 mM hydrogen peroxide, and different amounts of plasma, erythrocyte fraction, or liver mitochondria fraction
Determination of superoxide dismutase and SOD-mimetic activities
reproducible and require the use of the external reference sample. The SOD activity assay accuracy was estimated with the thermally inactivated biological samples, which had no statistically significant effect on chemiluminescence. The samples of the SOD were kept for 10 min at 95 °C for thermal inactivation.
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Conclusion The proposed model for the generation and registration of superoxide radical Co2+/hydrogen peroxide/lucigenin can be successfully used to determine the SOD activity in aqueous solutions, biological samples, as well as for comparison of the efficacy of SOD mimetics.
Determination of SOD activity in rat plasma, erythrocytes, and liver mitochondria References If the aim is the determination of SOD activity in cytoplasm or mitochondria (blood plasma or erythrocytes), the previous separation is needed prior to analysis. We used plasma, washed erythrocytes, and isolated mitochondria. Consequently, we could determine the activity of certain SOD isoforms in terms of the total protein. In the case of quantitation of total SOD, there is no need for separation. By using the CL method, the SOD activity was determined in rat blood plasma, erythrocytes, and mitochondria. The verification of results of the SOD activity quantitation in biological fluid samples was performed by the conventional procedure after Fridovich [1]. The main procedures of the sample preparation were similar to those recommended in reference [27]. To remove the interfering components (ascorbic acid, glutathione, catalase, and most other enzymes), the additional incubation of samples was performed at 60 °C during 10 min. The activity of SOD under thermal treatment reduces only at the temperature above 75 °C [17]. The data were plotted in A0/A versus Cp coordinates, where A0 was the intensity in the CL maximum in the absence of a sample; A was the intensity in the CL maximum in the presence of a plasma, erythrocyte, or mitochondria sample; and Cp is a protein concentration, in micrograms per milliliter (Fig. 2). The specific activities were found to be 11±1 units/mg of protein in plasma, 36± 2 units/mg of protein in erythrocytes, and 64±10 units/mg of protein in mitochondria. Indeed, the SOD activity of rat organs was different; the greatest activity was observed in liver and lesser ones where found in plasma and erythrocytes. This is in agreement with the other data [28]. The SOD activity assay accuracy was estimated with the thermally inactivated biological samples, which had no statistically significant effect on chemiluminescence. The samples of the specimens kept for 10 min at 95 °C for SOD thermal inactivation did not reduce the CL intensity statistically significant. So, we may conclude that the proposed procedure can be applied for the determination of SOD activity in different tissues. The SOD quantitation in real samples can be interfered by the presence of impurities such as cobalt or other transition metal catalysts, chelating ligands, antioxidant compounds, and other enzymes. In such cases, the standard addition analytical method may help.
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