Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase. Methods Enzymol 1999;301:169-77
Date
01/27/1999Pubmed ID
9919565DOI
10.1016/s0076-6879(99)01080-0Scopus ID
2-s2.0-0031795460 (requires institutional sign-in at Scopus site) 43 CitationsAbstract
NOS is a ubiquitous enzyme that has an oxygenase and reductase activity. NOS reduces electron acceptors, at the reductase domain, by a one-electron mechanism that is not inhibited by SOD. One example of this activity is the direct reduction of ferricytochrome c by nNOS. Redox cycling electron acceptors (EA in Scheme 1), such as lucigenin and NBT, are reduced by NOS to generate an intermediate radical (EAred). This radical can then be reoxidized to the parent compound by oxygen, and in the process generate superoxide. Consequently, both NBT and lucigenin will enhance NADPH-dependent superoxide generation in the presence of flavoprotein reductases such as NOS. The artificial generation of superoxide from lucigenin and NBT is a major pitfall in the use of these compounds as superoxide probes. We conclude that the use of ESR spin-trapping techniques, although not free of problems, is a viable technique for the detection and quantification of superoxide in systems containing nNOS.
Author List
Vásquez-Vivar J, Martásek P, Hogg N, Karoui H, Masters BS, Pritchard KA Jr, Kalyanaraman BAuthors
Neil Hogg PhD Senior Associate Dean, Professor in the Biophysics department at Medical College of WisconsinBalaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of Wisconsin
Kirkwood A. Pritchard PhD Professor in the Surgery department at Medical College of Wisconsin
Jeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
AcridinesAnimals
Electron Spin Resonance Spectroscopy
Free Radicals
Humans
Nitric Oxide Synthase
Spin Trapping
Superoxides
