Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching
Autor: | David Contreras, Victoria Melin, Juan David Figueroa, Eduardo Fuentes-Lemus, Beatriz Alvarez, Michael J. Davies, Eva Dorta, Mario Faúndez, Ana Denicola, Camilo López-Alarcón, Javiera Cortés-Ríos |
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Přispěvatelé: | Figueroa J.D., Fuentes-Lemus E., Dorta E., Melin V., Cortés-Rios J., Faúndez M., Contretas D., Denicola Ana, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica., Alvarez Beatriz, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica., Davies M.J., López-Alarcón C. |
Jazyk: | angličtina |
Rok vydání: | 2019 |
Předmět: |
Free Radicals
Peroxidase activity Bicarbonate Radical Clinical Biochemistry Carbonates Superoxide dismutase Carbonate radical anion Pyrogallol Biochemistry Pyrogallol red Bovine SOD-1 chemistry.chemical_compound Bleaching Agents Superoxide Dismutase-1 Spectrophotometry medicine ABTS Hydrogen peroxide lcsh:QH301-705.5 Human SOD-1 lcsh:R5-920 biology medicine.diagnostic_test Spin trapping DMPO Spectrum Analysis Organic Chemistry Method Article Bicarbonates lcsh:Biology (General) chemistry biology.protein EPR lcsh:Medicine (General) Oxidation-Reduction Peroxidase Nuclear chemistry |
Zdroj: | David Figueroa, J, Fuentes-Lemus, E, Dorta, E, Melin, V, Cortes-Rios, J, Faundez, M, Contreras, D, Denicola, A, Alvarez, B, Davies, M J & Lopez-Alarcon, C 2019, ' Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching ', Redox Biology, vol. 24, 101207 . https://doi.org/10.1016/j.redox.2019.101207 COLIBRI Universidad de la República instacron:Universidad de la República Redox Biology, Vol 24, Iss, Pp-(2019) |
Popis: | Carbonate radicals (CO3·-) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO3·- formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared to previously reported methods using electron paramagnetic resonance spin trapping with DMPO, and the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid). The kinetics of PGR consumption elicited by CO3·- was followed by visible spectrophotometry. Solutions containing PGR (5–200 μM), SOD-1 (0.3–3 μM), H2O2 (2 mM) in bicarbonate buffer (200 mM, pH 7.4) showed a rapid loss of the PGR absorption band centered at 540 nm. The initial consumption rate (Ri) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3·- release of 24.6 ± 4.3 μM min−1 for 3 μM bSOD-1. Both bSOD-1 and hSOD-1 showed a similar peroxidase activity, with enzymatic inactivation occurring over a period of 20 min. The single Trp residue (Trp32) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 ± 0.1 μM min−1) with this occurring more rapidly than hSOD-1 inactivation, suggesting that these processes are not directly related. Added free Trp was rapidly oxidized in competition with PGR. These data indicate that PGR reacts rapidly and efficiently with CO3·- resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3·- release from bSOD-1 and hSOD-1 peroxidase activity. Keywords: Carbonate radical anion, Superoxide dismutase, Hydrogen peroxide, Pyrogallol red, Peroxidase activity, Human SOD-1, Bovine SOD-1, EPR, DMPO, ABTS |
Databáze: | OpenAIRE |
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