Detection of superoxide ions

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 from either the xanthine-xanthine oxidase reaction or by the addition of 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±0.3×105 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 2-200 μM KO2 while 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 to some other commonly used methods for detection of superoxide (i.e. nitroblue tetrazolium). Results presented showed good experimental agreements with rates obtained for the measurement of superoxide ion when compared to other well known probes such as acetylated ferri cytochrome-C and XTT. A detailed discussion of the advantages and limitations of this new superoxide ion probe is presented.

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