Activated oxygen generation from thylakoids: A novel spin trap

Using a novel phosphorylated spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-p yrroline-N-oxide (DEPMPO), an analogue of the commonly used spin trap 5,5 ' -dimethyl-1-pyrroline N-oxide (DMPO), we have investigated the production o f oxygen radical species under illumination of thylakoids from wheat (Triti cum durum Desf co. Ofanto). DEPMPO reacted with superoxide (O2(.-)) and hyd roxyl radical (HO.) forming distinctive spin trap adducts. Spectra of (O-2( .-)) and HO. adducts of DEPMPO were recorded in the presence of xanthine/xa nthine oxidase and FeSO4/H2O2, respectively, and computer simulation of spe ctra was performed. During illumination of thylakoids both O-2(.-) and HO. were detected as well. Transition metals catalysed transformation of O-2(.- ) into HO.. The conversion was enhanced by H2O2 and prevented by exogenous superoxide dismutase and catalase. The presence of a thylakoid-bound supero xide dismutase, whose activity was inhibited by H2O2 and diethyl-dithiocarb amic acid, was responsible for H2O2 production from O-2(.-) and thus for HO . generation.