The amplex red/horseradish peroxidase assay requires superoxide dismutase to measure hydrogen peroxide in the presence of NAD(P)H

Abstract

A sensitive fluorescence assay based on Amplex Red (AR) oxidation by horseradish peroxidase (AR/HRP) is described which continuously monitor rates of H 2 O 2 production by microsomal enzymes in the presence of relatively high concentrations of NADPH. NADPH and NADH are known to interact with HRP and generate significant quantities of superoxide anion, a radical that spontaneously dismutates to form H 2 O 2 which interferes with the AR/HRP assay. Microsomal enzymes generate H 2 O 2 as a consequence of electron transfer from NADPH to cytochrome P450 hemoproteins with subsequent oxygen activation. We found that superoxide anion formation via the interaction of NADPH with HRP was inhibited by superoxide dismutase (SOD) without affecting H 2 O 2 generation by microsomal enzymes. Using SOD in enzyme assays, we consistently detected rates of H 2 O 2 production using microgram quantities of microsomal proteins (2.62 ± 0.20 picomol/min/μg protein for liver microsomes from naïve female rats, 12.27 ± 1.29 for liver microsomes from dexamethasone induced male rats, and 2.17 ± 0.25 picomol/min/μg protein for human liver microsomes). This method can also be applied to quantify rates of H 2 O 2 production by oxidases where superoxide anion generation by NADH or NADPH and HRP can interfere with enzyme assays.

Tissue‐specific differences in Ca2+ sensitivity of the mitochondrial permeability transition pore (PTP). Experiments in male rat liver and heart

“…Fluorescence was measured at 571 and 585 nm in a POLARstar Omega detector (BGM LABTECH, Offenburg, Germany). Results were interpolated against a calibration curve (0–200 nM H 2 O 2 ) to convert relative fluorescence units (RFU) to nm H 2 O 2 (Mishin et al., 2020 ).…”

Exploring the partial degradation of polysaccharides: Structure, mechanism, bioactivities, and perspectives

“…The mechanism of radical cleavage of polysaccharides: (a) free radical formation induced by the addition of metal ions (Chen, Sun‐Waterhouse, et al., 2021); (b) free radical formation induced by the addition of ascorbic acid (Zou et al., 2020); (c) free radical formation in an enzyme–H 2 O 2 system (Mishin et al., 2020); (d) free radical formation induced by ultrasound (Rahdar et al., 2019); (e) free radical formation induced by UV radiation (Chen, Cai et al., 2018); (f) free radical formation induced by plasma radiation (Wu et al., 2018); free radical degradation of (g) chondroitin sulfate and (h) NS‐heparosan (Zhang et al., 2020). (Copyright © 2020 Elsevier B.V. All rights reserved, reprinted with permission)…”

Tissue-specific differences in Ca 2+ sensitivity of the mitochondrial permeability transition pore. Experiments in rat liver and heart

“…Fluorescence was measured at 571 and 585 nm in a POLARstar Omega detector (BGM LABTECH, Offenburg, Germany). Results were interpolated against a calibration curve (0-200 nM H 2 O 2 ) to convert relative uorescence units (RFU) to nm H 2 O 2 (Mishin et al, 2020). Statistical analysis.…”