Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry Vijay Kumar Kutala, Narasimham L. Parinandi, Jay L. Zweier, and Periannan Kuppusamy * Center for Biomedical EPR Spectroscopy and Imaging, Department of Internal Medicine, Davis Heart and Lung Research Institute, Ohio State University, Columbus, OH, USA Received 16 December 2003, and in revised form 20 January 2004 Abstract Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of speciﬁc compounds. The redox methods, however, suﬀer from various interferences, which limit their use in the assay of superoxide. Electron para- magnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1  10 3 M 1 s 1 . In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the re- action between TAM and superoxide. The eﬀects of diﬀerent ﬂuxes of superoxide and concentrations of TAM on the eﬃciency and sensitivity of quantiﬁcation of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of su- peroxide and further revealed that the assay is not aﬀected by the presence of hydrogen peroxide. In summary, the TAM spec- trophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Trityl radical; Spectrophotometry; Superoxide; Xanthine oxidase; Cytochrome c Measurement of the production of superoxide (O Å 2 ) and other oxygen-derived free radicals is of utmost im- portance in the study of numerous physiological and pathological processes. A variety of methods have been developed to detect and quantify superoxide radicals in biological systems. These include spectrophotometric methods that use reduction of ferricytochrome c or ni- troblue tetrazolium (NBT) by superoxide, chemilumi- nescence, ﬂuorescence-based techniques, and electron paramagnetic resonance (EPR) 1 spectroscopy [1–5]. Among these, the most widely used cytochrome c re- duction and lucigenin-ampliﬁed chemiluminescence as- says are considered as reliable methods for the determination of superoxide. EPR spin-trapping, how- ever, remains the most direct and deﬁnitive method for the detection of superoxide radicals. Barbacanne et al. [6] compared various methods of detection of superox- ide in cellular system and suggested that ferricytochrome c reduction, EPR spectroscopy, and hydroethi- dine-based ﬂuorescence techniques are suitable for measuring extracellular superoxide generation. The 12-myristate-13-acetate; PMN, polymorphonuclear neutrophil; SOD, superoxide dismutase; TAM, triarylmethyl; X/XO, xanthine/xanthine oxidase. * Corresponding author. Fax: 1-614-292-8454. E-mail address: kuppusamy-1@medctr.osu.edu (P. Kuppusamy). 1 Abbreviations used: DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DTPA, diethylenetriaminepentaacetic acid; EPR, electron paramag- netic resonance; LRED, light/riboﬂavin/electron/donor; PMA, phorbol- 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.01.020 Archives of Biochemistry and Biophysics 424 (2004) 81–88 ABB www.elsevier.com/locate/yabbi