In the present work we show that structural and kinetic information can be obtained from the paramagnetic resonance spectrum of a rather simple stable radical chemically bonded to biomolecules. Paramagnetic nitroxide radicals colntaining the group R R Me> jMe Me N-Me are remarkably stable and inert,1 and show sharp, well-resolved, and simple paramagnetic resonance spectra2 that are sensitive to molecular motion,3 and, to a lesser extent, sensitive to polarity of the molecular environment.4 As shown below, such nitroxide radicals containing an isocyanate group can be bound to proteins and synthetic polypeptides in a manner similar to that used previously to attach fluorescent dyes.5 Here we report a preliminary study of the paramagnetic resoiiance of a nitroxide radical bonded to bovine serum albumin and to poly-L-lysine. It is evident from the present study that there are many other potential applicatioiis of this method to biological systems.
The effects of intracellularly generated H2O2 on cell viability, morphology, and biochemical markers of injury have been investigated in a clonal cell line of neuronal origin (140-3, mouse neuroblastoma X rat glioma) as a cell culture model for the role of oxidative stress in the long-term loss of neurons in the brain. The H2O2 was generated from the redox cycling of menadione, or by the oxidation of serotonin catalyzed by monoamine oxidase, to simulate the effect of amine neurotransmitter turnover. Incubation with menadione at concentrations as low as 10 microM for several hours resulted in significant losses of cell viability and altered morphology. Similar effects were evident in the presence of serotonin only after incubation overnight with concentrations > 1 mM. The cytotoxicity of either agent was potentiated by preincubation with specific inhibitors of two enzymes important to cellular antioxidant defenses, 3-amino-1,2,4-triazole for catalase and 1,3-bis(chloromethyl)-1-nitrosourea for glutathione reductase. Activity of another antioxidant enzyme of particular importance to antioxidant defenses in brain, the selenoprotein glutathione peroxidase, was stimulated fourfold by growth of cultures in the presence of sodium selenite as a source of active-site Se for the enzyme. The only effect of the selenite on other functionally coupled antioxidant enzymes was a decrease in activity of superoxide dismutase at concentrations > 200 nM. The selenite substantially protected cells against oxidative stress induced by combinations of menadione, 3-amino-1,2,4-triazole, and 1,3-bis(chloromethyl)-1-nitrosourea, but was only marginally effective with serotonin as a source of oxidative stress. The monoamine oxidase inhibitor pargyline increased cell survival in the presence of serotonin, demonstrating the role of this enzyme in its cytotoxicity. DNA damage (single strand breaks), but not lipid peroxidation, correlated with the cytotoxic effects of menadione.
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