Superoxide dismutase versus ferricytochrome C: determining rate constants for the spin trapping of superoxide by cyclic nitrones.
Given that spin trapping/electron paramagnetic resonance (EPR) spectroscopy has become the primary technique to identify important biologically generated free radicals, such as superoxide (O(2)(*-)), in vitro and in vivo models, evaluation of the efficiency of specific spin traps to identify this free radical is paramount. Recently, a family of ester-containing nitrones has been prepared, which appears to have distinct advantages for spin trapping O(2)(*-) compared to the well-studied spin traps 5,5-dimethyl-1-pyrroline N-oxide 1 and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide 2. An important determinant in the selection of a spin trap is the rate constant (k(app)) for its reaction with O(2)(*-), and several different methods have been employed in estimating this k(app). In this paper, the two most frequently used scavengers of O(2)(*-), ferricytochrome c and Cu/Zn-SOD, were evaluated as competitive inhibitors for spin trapping this free radical. Data presented herein demonstrate that SOD is the preferred compound when determining the k(app) for the reaction of O(2)(*-) with spin traps. Using this model, the k(app) for the reaction of nitrone 1, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 3, and 5-methoxycarbonyl-5-methyl-1-pyrroline N-oxide 4 with O(2)(*)(-) was estimated to be 24.6 +/- 3.1, 73.0 +/- 12, and 89.4 +/- 1.0 M(-1) s(-1) at pH 7.0, respectively. Several other comparative studies between known spin traps were also undertaken.
Weaver
John
J
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA.
Tsai
Pei
P
Pou
Sovitj
S
Rosen
Gerald M
GM
D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D009589:Nitrogen Oxides / Q000032:analysis / Q000737:chemistry*; D019097:Spin Trapping / Q000379:methods*; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D013481:Superoxides / Q000032:analysis / Q000737:chemistry*
D009589:Nitrogen Oxides; C015388:nitrones; D013481:Superoxides; D045304:Cytochromes c; D013482:Superoxide Dismutase