TY - JOUR

T1 - Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching

AU - David Figueroa, Juan

AU - Fuentes-Lemus, Eduardo

AU - Dorta, Eva

AU - Melin, Victoria

AU - Cortes-Rios, Javiera

AU - Faundez, Mario

AU - Contreras, David

AU - Denicola, Ana

AU - Alvarez, Beatriz

AU - Davies, Michael J.

AU - Lopez-Alarcon, Camilo

PY - 2019

Y1 - 2019

N2 - Carbonate radicals (CO3 center 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 CO3 center 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 CO3 center dot(-) was followed by visible spectrophotometry. Solutions containing PGR (5-200 mu M), SOD-1 (0.3-3 mu M), 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 (R-i) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3 center dot(-) release of 24.6 +/- 4.3 mu M min(-1) for 3 mu M 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 (Trp(32)) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 +/- 0.1 mu M 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 center dot(-) resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3 center dot(-)release from bSOD-1 and hSOD-1 peroxidase activity.

AB - Carbonate radicals (CO3 center 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 CO3 center 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 CO3 center dot(-) was followed by visible spectrophotometry. Solutions containing PGR (5-200 mu M), SOD-1 (0.3-3 mu M), 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 (R-i) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3 center dot(-) release of 24.6 +/- 4.3 mu M min(-1) for 3 mu M 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 (Trp(32)) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 +/- 0.1 mu M 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 center dot(-) resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3 center dot(-)release from bSOD-1 and hSOD-1 peroxidase activity.

KW - Carbonate radical anion

KW - Superoxide dismutase

KW - Hydrogen peroxide

KW - Pyrogallol red

KW - Peroxidase activity

KW - Human SOD-1

KW - Bovine SOD-1

KW - EPR

KW - DMPO

KW - ABTS

U2 - 10.1016/j.redox.2019.101207

DO - 10.1016/j.redox.2019.101207

M3 - Journal article

C2 - 31102971

VL - 24

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

M1 - 101207

ER -