Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole

Olojo, R.O.; Xia, R.H.; Abramson, J.J.

doi:10.1016/j.ab.2005.01.032

Cited by 136Redox biology and oxidative stressAntioxidant Activity and Oxidative StressMolecular Sensors and Ion Detection

Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole

Olojo, R.O., Xia, R.H., Abramson, J.J.·2005·Analytical Biochemistry

DOI 10.1016/j.ab.2005.01.032

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Abstract

Two highly sensitive spectrophotometric methods are developed and described for the measurement of superoxide ion radical derived from KO2 as well as O2*- generated either from the xanthine-xanthine oxidase reaction or by the addition of nicotinamide adenine dinucleotide (NADH) to skeletal muscle sarcoplasmic reticulum (SR) vesicles. These methods allow quantification of superoxide ion concentration by monitoring its reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), either by recording absorbance of the final reaction product at a wavelength of 470 nm or by measuring its fluorescence emission intensity at 550 nm using an excitation wavelength of 470 nm. The extinction coefficient of the active product was determined to be 4000 M(-1) cm(-1). A lower limit second-order bimolecular rate constant of 1.5+/-0.3x10(5) M(-1) s(-1) was estimated from kinetic stopped-flow analysis for the reaction between NBD-Cl and KO2. A plot of absorbance versus concentration of superoxide was linear over the range 2 to 200 microM KO2, whereas higher sensitivities were obtained from fluorometric measurements down into sub-micromolar concentrations with a limit of detection of 100 nM KO2. This new spectrophotometric assay showed higher specificity when compared with some other commonly used methods for detection of superoxide (e.g., nitroblue tetrazolium). Results presented showed good experimental agreement with rates obtained for the measurement of superoxide ion when compared with other well-known probes such as acetylated ferri cytochrome c and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT). A detailed discussion of the advantages and limitations of this new superoxide ion probe is presented.

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The 4-chloro-7-nitrobenzo [c] (1,2,5) oxadiazole probe is commercially available and is used for rapid detection and quantification of superoxide radical generated by the xanthine/xanthine oxidase system.
“The 4-chloro-7-nitrobenzo [c] (1,2,5) oxadiazole probe is commercially available and is used for the rapid detection and quantification of the superoxide radical generated by the xanthine/xanthine oxidase system [162].”
From Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity (2023) · cites this paper as [162]

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Authors: Olojo, R. O., Xia, R. H., & Abramson, J. J.
Affiliations: Portland State University (US)
East China Normal University (CN)
Corresponding author: Abramson, J.J.
ORCID: Xia, R.H.
Venue: Analytical Biochemistry
Publisher: Elsevier BV
Published: 2005-04
Volume: 339
Issue: 2
Pages: 338-344
ISSN: 0003-2697 (ISSN-L)
DOI: 10.1016/j.ab.2005.01.032
OpenAlex: W1963848156
Cited by: 136
References: 27
Citations by year: 2026: 11 · 2025: 6 · 2024: 12 · 2023: 10 · 2022: 16 · 2021: 1 · 2020: 8 · 2019: 7 · 2018: 2 · 2017: 14 · 2016: 7 · 2015: 11 · 2014: 3 · 2013: 3 · 2012: 8
FWCI: 0.63
Type: journal article
Language: en
Open access: closed
Licence: www.elsevier.com/tdm/userlicense/1.0/ (tdm)
www.elsevier.com/legal/tdmrep-license (tdm)
Locations: Analytical Biochemistry (publishedVersion)
PubMed (publishedVersion)
Funders: National Institutes of Health
National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01-AR48911, R01AR048911)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
Physical Sciences › Chemistry › Spectroscopy › Molecular Sensors and Ion Detection
Keywords: superoxide, NBD-Cl, spectrophotometric assay, fluorometric assay, NADH, sarcoplasmic reticulum vesicles, potassium superoxide, reaction kinetics
MeSH terms: Animals, Fluorometry, 4-Chloro-7-nitrobenzofurazan, Rabbits, Sarcoplasmic Reticulum, Sensitivity and Specificity, Spectrophotometry, Superoxides, Xanthine Oxidase, Reproducibility of Results, Xanthine

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Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole

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