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Enhanced superoxide and hydrogen peroxide detection in biological assays
SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS João V. Rodrigues, Cláudio M. Gomes
Table S1 – Comparison of different methods for superoxide quantification ‐1 ‐1
Compound SOR/SOD
k (M s ) 9 ~1×10
XTT MTS NBT Cytochrome c Dihidroethidine/Mitosox
8.6 ×10 5 1.3×10 4 6 ×10 5 2.6×10 6 2‐4×10
4
Method - Fluorescence (λex = 563 nm, λem =587 nm) ‐1 ‐1 - Visible absorption (λ= 571 nm, εapp = 24.000 cm M ) ‐1 ‐1 ‐ Visible absorption (λ= 490 nm, ε = 26.900 cm M ) ‐1 ‐1 ‐ Visible absorption (λ= 470 nm, ε = 21.600 cm M ) ‐1 ‐1 ‐ Visible absorption ((λ= 530 nm, ε = 12.800 cm M ) ‐1 ‐1 ‐ Visible absorption (λ= 550 nm, ε = 21.000 cm M ) - Fluorescence
Reference This work [1] [1] [2] [3] [4, 5]
[1] Sutherland, M.W. and B.A. Learmonth, The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase. Free Radic Res, 1997. 27(3): p. 283‐9. [2] Bielski, B.H.J., G.G. Shiue, and S. Bajuk, Reduction of nitro tetrazolium blue by CO−2 and O−2 radicals. Journal of Physical Chemistry 1980. 84: p. 830‐833. [3] Butler, J., W.H. Koppenol, and E. Margoliash, Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion. J Biol Chem, 1982. 257(18): p. 10747‐50. [4] Robinson, K.M., et al., Selective fluorescent imaging of superoxide in vivo using ethidium‐based probes. Proc Natl Acad Sci U S A, 2006. 103(41): p. 15038‐43. [5] Zielonka, J., et al., Pulse radiolysis and steady‐state analyses of the reaction between hydroethidine and superoxide and other oxidants. Arch. Biochem. Biophys., 2006. 456: p. 39‐47
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SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS João V. Rodrigues, Cláudio M. Gomes
1.2
Resorufin (μM)
1.0 0.8 0.6 0.4 0.2 0.0 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Hydrogen Peroxide (μM) Figure S1 – Effect of reduced SOR on the calibration curves for H2O2-dependent resorufin formation. Reaction media contained 23 µM AR, 2.2 U/mL HRP and 2 mM Tris pH 8.0, and the resorufin concentration was determined by measuring the absorbance at 571 nm after each H2O2 addition. Experiments were performed in the absence (closed circles) and the presence of 0.5 µM SOR (open circles) and 0.9 µM SOR (closed squares), and the presence of 0.5 µM SOD (open squares).
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SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS
Oxidized product (μM)
João V. Rodrigues, Cláudio M. Gomes
1.0 0.8 0.6 0.4 0.2 0.0 0.0
0.5
1.0
1.5
2.0
2.5
Hydrogen peroxide (μM) Figure S2. Effect of SOR and ascorbate on the oxidation of AR to resorufin by H2O2. The reaction media contained 1 µM AR and 0.4 U/mL HRP in 2 mM Tris pH 8.0 and the formation of resorufin was monitored at 571 nm after each H2O2 addition. The experiments were performed in the absence (closed squares) and in the presence of 0.4 µM SOR (open circles) and 0.8 µM SOR (closed circles), or 1 µM Ascorbate (open triangles). Note: Upon full depletion of all Amplex Red, the accumulated resorufin product starts to be a substrate of HRP being oxidized by further additions of H2O2 (Reszka, K. J., Wagner, B. A., Burns, C. P. and Britigan, B. E., Effects of peroxidase substrates on the Amplex red/peroxidase assay: antioxidant properties of anthracyclines. Anal Biochem 2005, 342, (2), 327-37.), and this is evidenced by the decrease in absorbance at 571 nm after 1:1 oxidation of Amplex red. This reaction is not relevant in our H2O2-quantification assay since Amplex Red (23 μM) is in large excess over the maximal H2O2 concentration that is measured in our assay conditions (typically below 4 μM).
3/5
SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS João V. Rodrigues, Cláudio M. Gomes
600
-1
H2O2 (nM min ) w/SOR
800
400
200
0
0
200
400
600
800
-1 H2O2 (nM.min ) w/SOD
Figure S3 - Comparison of the detection of H2O2 by the AR/HRP system supplemented with ascorbate in the presence of SOD or SOR. Glucose (1mM) and different concentrations of glucose oxidase were used to generate a constant flux of H2O2, which was detected by the monitoring the oxidation of AR at 571 nm in the presence of 2.2 U/mL HRP, 1 mM EDTA, 8 µM ascorbate, and either 0.5 µM SOD or 0.5 µM SOR. The extinction coefficient of AR used was 24000 M-1 cm-1. The solid line represents the linear fit to the data points (slope = 1.05 ± 0.06, intercept = 40 ± 20 nM/min).
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SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS João V. Rodrigues, Cláudio M. Gomes
a
-1
H2O2 (nM.min )
1600 1200 800 400
SOR SOD
0
b
-1
O2 (nM.min )
800 600
α = 0.96
-
400 200 0
0
200
400
600
800
1000 -1
Cytochrome c reduction (nM.min ) Figure S4- Superoxide detection by the AR/HPR/SOR assay monitored by visible absorption at 571 nm. (a) Presence of 0.5 µM SOR increases the rate of H2O2 formation, in comparison with 0.5 µM SOD, as measured at different O2•- fluxes calibrated by the cytochrome c reduction assay. Solid lines represent linear fits to the data (SOR slope= 1.92 ± 0.05, intercept = 20 ± 20; SOD slope = 1.44 ± 0.06, intercept = 0 ± 30). (b) Comparison of superoxide detected by the AR/HRP/SOR assay versus the standard cytochrome c reduction assay. The solid line represents the linear fit to the data (slope = 0.96 ± 0.2, intercept = 30 ± 30 nM/min).
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Table S1 – Comparison of different methods for superoxide quantification ‐1 ‐1
Compound SOR/SOD
k (M s ) 9 ~1×10
XTT MTS NBT Cytochrome c Dihidroethidine/Mitosox
8.6 ×10 5 1.3×10 4 6 ×10 5 2.6×10 6 2‐4×10
4
Method - Fluorescence (λex = 563 nm, λem =587 nm) ‐1 ‐1 - Visible absorption (λ= 571 nm, εapp = 24.000 cm M ) ‐1 ‐1 ‐ Visible absorption (λ= 490 nm, ε = 26.900 cm M ) ‐1 ‐1 ‐ Visible absorption (λ= 470 nm, ε = 21.600 cm M ) ‐1 ‐1 ‐ Visible absorption ((λ= 530 nm, ε = 12.800 cm M ) ‐1 ‐1 ‐ Visible absorption (λ= 550 nm, ε = 21.000 cm M ) - Fluorescence
Reference This work [1] [1] [2] [3] [4, 5]
[1] Sutherland, M.W. and B.A. Learmonth, The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase. Free Radic Res, 1997. 27(3): p. 283‐9. [2] Bielski, B.H.J., G.G. Shiue, and S. Bajuk, Reduction of nitro tetrazolium blue by CO−2 and O−2 radicals. Journal of Physical Chemistry 1980. 84: p. 830‐833. [3] Butler, J., W.H. Koppenol, and E. Margoliash, Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion. J Biol Chem, 1982. 257(18): p. 10747‐50. [4] Robinson, K.M., et al., Selective fluorescent imaging of superoxide in vivo using ethidium‐based probes. Proc Natl Acad Sci U S A, 2006. 103(41): p. 15038‐43. [5] Zielonka, J., et al., Pulse radiolysis and steady‐state analyses of the reaction between hydroethidine and superoxide and other oxidants. Arch. Biochem. Biophys., 2006. 456: p. 39‐47
1/5
SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS João V. Rodrigues, Cláudio M. Gomes
1.2
Resorufin (μM)
1.0 0.8 0.6 0.4 0.2 0.0 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Hydrogen Peroxide (μM) Figure S1 – Effect of reduced SOR on the calibration curves for H2O2-dependent resorufin formation. Reaction media contained 23 µM AR, 2.2 U/mL HRP and 2 mM Tris pH 8.0, and the resorufin concentration was determined by measuring the absorbance at 571 nm after each H2O2 addition. Experiments were performed in the absence (closed circles) and the presence of 0.5 µM SOR (open circles) and 0.9 µM SOR (closed squares), and the presence of 0.5 µM SOD (open squares).
2/5
SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS
Oxidized product (μM)
João V. Rodrigues, Cláudio M. Gomes
1.0 0.8 0.6 0.4 0.2 0.0 0.0
0.5
1.0
1.5
2.0
2.5
Hydrogen peroxide (μM) Figure S2. Effect of SOR and ascorbate on the oxidation of AR to resorufin by H2O2. The reaction media contained 1 µM AR and 0.4 U/mL HRP in 2 mM Tris pH 8.0 and the formation of resorufin was monitored at 571 nm after each H2O2 addition. The experiments were performed in the absence (closed squares) and in the presence of 0.4 µM SOR (open circles) and 0.8 µM SOR (closed circles), or 1 µM Ascorbate (open triangles). Note: Upon full depletion of all Amplex Red, the accumulated resorufin product starts to be a substrate of HRP being oxidized by further additions of H2O2 (Reszka, K. J., Wagner, B. A., Burns, C. P. and Britigan, B. E., Effects of peroxidase substrates on the Amplex red/peroxidase assay: antioxidant properties of anthracyclines. Anal Biochem 2005, 342, (2), 327-37.), and this is evidenced by the decrease in absorbance at 571 nm after 1:1 oxidation of Amplex red. This reaction is not relevant in our H2O2-quantification assay since Amplex Red (23 μM) is in large excess over the maximal H2O2 concentration that is measured in our assay conditions (typically below 4 μM).
3/5
SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS João V. Rodrigues, Cláudio M. Gomes
600
-1
H2O2 (nM min ) w/SOR
800
400
200
0
0
200
400
600
800
-1 H2O2 (nM.min ) w/SOD
Figure S3 - Comparison of the detection of H2O2 by the AR/HRP system supplemented with ascorbate in the presence of SOD or SOR. Glucose (1mM) and different concentrations of glucose oxidase were used to generate a constant flux of H2O2, which was detected by the monitoring the oxidation of AR at 571 nm in the presence of 2.2 U/mL HRP, 1 mM EDTA, 8 µM ascorbate, and either 0.5 µM SOD or 0.5 µM SOR. The extinction coefficient of AR used was 24000 M-1 cm-1. The solid line represents the linear fit to the data points (slope = 1.05 ± 0.06, intercept = 40 ± 20 nM/min).
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SUPPLEMENTARY MATERIALS ENHANCED SUPEROXIDE AND HYDROGEN PEROXIDE DETECTION IN BIOLOGICAL ASSAYS João V. Rodrigues, Cláudio M. Gomes
a
-1
H2O2 (nM.min )
1600 1200 800 400
SOR SOD
0
b
-1
O2 (nM.min )
800 600
α = 0.96
-
400 200 0
0
200
400
600
800
1000 -1
Cytochrome c reduction (nM.min ) Figure S4- Superoxide detection by the AR/HPR/SOR assay monitored by visible absorption at 571 nm. (a) Presence of 0.5 µM SOR increases the rate of H2O2 formation, in comparison with 0.5 µM SOD, as measured at different O2•- fluxes calibrated by the cytochrome c reduction assay. Solid lines represent linear fits to the data (SOR slope= 1.92 ± 0.05, intercept = 20 ± 20; SOD slope = 1.44 ± 0.06, intercept = 0 ± 30). (b) Comparison of superoxide detected by the AR/HRP/SOR assay versus the standard cytochrome c reduction assay. The solid line represents the linear fit to the data (slope = 0.96 ± 0.2, intercept = 30 ± 30 nM/min).
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