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

Two highly sensitive spectrophotometric methods are developed and described for the measurement of superoxide ion radical derived from KO2 Lis well as O-2(.-) 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 super-oxide ion concentration by monitoring its reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-C1), either by recording absorbance of the final reaction product at a wavelength of 470 run or by measuring its fluorescence emission intensity at 550 run using an excitation wavelength of 470 urn. 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 x 10(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 mu M KO2, whereas higher sensitivities were obtained from fluorometric measurements down into submicromolar concentrations with a limit of detection of 100 it 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. (c) 2005 Elsevier Inc. All rights reserved.