An EPR spin-probe and spin-trap study of the free radicals produced by plant plasma membranes

Plant plasma membranes are known to produce superoxide radicals, while the production of hydroxyl radical is thought to occur only in the cell wall. In this work it was demonstrated using combined spin-trap and spin-probe EPR spectro- scopic techniques, that plant plasma membranes do produce superoxide and hydro- xyl radicals but by kinetically different mechanisms. The results show that superoxide and hydroxyl radicals can be detected by DMPO spin-trap and that the mechanisms and location of their production can be differentiated using the reduction of spin-pro- bes Tempone and 7-DS. It was shown that the mechanism of production of oxygen reactive species is NADH dependent and diphenylene iodonium inhibited. The ki- netics of the reduction of Tempone, combined with scavengers or the absence of NADH indicates that hydroxyl radicals are produced by a mechanism independent of that of superoxide production. It was shown that a combination of the spin-probe and spin-trap technique can be used in free radical studies of biological systems, with a number of advantages inherent to them.