Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching

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dc.article.number101207
dc.catalogadorgjm
dc.contributor.authorFigueroa Alegría, Juan David
dc.contributor.authorFuentes Lemus, Eduardo Felipe
dc.contributor.authorDorta Pérez, Eva
dc.contributor.authorMelin, Victoria
dc.contributor.authorCortés Ríos, Javiera Alejandra
dc.contributor.authorFaúndez Cáceres, Mario
dc.contributor.authorContreras, David
dc.contributor.authorDenicola, Ana
dc.contributor.authorÁlvarez, Beatriz
dc.contributor.authorDavies, Michael J.
dc.contributor.authorLópez Alarcón, Camilo Ignacio
dc.date.accessioned2024-12-30T15:01:37Z
dc.date.available2024-12-30T15:01:37Z
dc.date.issued2019
dc.description.abstractCarbonate radicals (CO3radical dot-) 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 CO3radical dot- 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 CO3radical dot- 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 CO3radical dot- 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 CO3radical dot- resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3radical dot- release from bSOD-1 and hSOD-1 peroxidase activity.
dc.description.funderVRAID (ex DIPUC)
dc.fechaingreso.objetodigital2024-12-30
dc.format.extent9 páginas
dc.fuente.origenHistorial Académico
dc.identifier.citationFigueroa Alegria Juan David, Fuentes Lemus Eduardo Felipe, Faundez Caceres Mario Antonio, Lopez Alarcon Camilo Ignacio. Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching. 2019;24:1-9.
dc.identifier.doi10.1016/j.redox.2019.101207
dc.identifier.issn2213-2317
dc.identifier.urihttps://doi.org/10.1016/j.redox.2019.101207
dc.identifier.urihttps://repositorio.uc.cl/handle/11534/89365
dc.identifier.wosidWOS:000471255400038
dc.information.autorucEscuela de Química; Figueroa Alegría, Juan David; S/I; 1071087
dc.information.autorucEscuela de Química; Fuentes Lemus, Eduardo Felipe; 0000-0002-1465-8466; 186720
dc.information.autorucEscuela de Química; Dorta Pérez, Eva; 0000-0002-2292-0356; 237939
dc.information.autorucEscuela de Ingeniería; Cortés Ríos, Javiera Alejandra; S/I; 1092020
dc.information.autorucEscuela de Química; Faúndez Cáceres, Mario; 0000-0002-5507-3579; 174901
dc.information.autorucEscuela de Química; López Alarcón, Camilo Ignacio; S/I; 1004308
dc.language.isoen
dc.nota.accesocontenido completo
dc.revistaRedox Biology
dc.rightsacceso abierto
dc.rights.licenseCC BY-NC-ND 4.0 Attribution-NonCommercial-NoDerivatives 4.0 International Deed
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectCarbonate radical anion
dc.subjectSuperoxide dismutase
dc.subjectHydrogen peroxide
dc.subjectPyrogallol red
dc.subjectPeroxidase activity
dc.subjectHuman SOD-1
dc.subjectBovine SOD-1
dc.subjectEPR
dc.subjectDMPO
dc.subjectABTS
dc.subject.ddc510
dc.subject.deweyMatemática física y químicaes_ES
dc.subject.ods03 Good health and well-being
dc.subject.odspa03 Salud y bienestar
dc.titleQuantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching
dc.typeartículo
dc.volumen24
sipa.codpersvinculados1071087
sipa.codpersvinculados186720
sipa.codpersvinculados237939
sipa.codpersvinculados1092020
sipa.codpersvinculados174901
sipa.codpersvinculados1004308
sipa.trazabilidadHistorial Académico;09-07-2021
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