Abstract 407 Neonatal Disease Oriented Research: Molecular Events and Brain Injury Poster Symposium, Tuesday, 5/4

Reactive oxygen species have been implicated in many diseases, particularly in prematurely born infants who have underdeveloped antioxidant defense capabilities. Although oxidant changes in glutathione (GSH) and glutathione disulfide (GSSG) levels have been studied, adverse effects associated with changes in thiol/disulfide status probably are mediated by changes in protein thiol (PSH) status. Decreases in total cellular PSH have been reported from studies of oxidant challenge in vitro, but we have not observed depletion of PSH in models of oxidant injury in vivo. To test the hypothesis that oxidant shifts in thiol status are expressed preferentially on individual proteins, we treated tissue samples with the thiol-reactive reagent monobromobimane (mBBr), separated the derivatized proteins by SDS-PAGE, and visualized the highly fluorescent thioether derivatives. Mice treated with hepatotoxic doses of acetaminophen (AP) show selective losses of PSH status and enzyme activity, but not of protein content, of the mitochondrial enzyme carbamyl phosphate-I. Incubation of mitochondrial fractions of AP-treated mice with dithiothreitol (DTT) or other disulfide-reducing agents failed to restore mBBr-derived fluorescence of CPS-I, suggesting that formation of protein disulfides PSSG or PSSP were unlikely. Incubation of mitochondrial fractions from control mice with GSSG in vitro prior to derivatization of PSH with mBBr decreased fluorescence intensities in a dose-dependent manner. CPS-I was not affected preferentially, in further support of the hypothesis that S-thiolations are not responsible for the effects of AP in vivo. However, treatment with DTT after incubation with GSSG was surprisingly ineffective in restoring mBBr-dependent fluorescence. Exposure of tissue fractions to HOCl in vitro, as a potential model of the molecular effects of inflammation and activation of myeloperoxidase in vivo, also diminished mBBr-derived fluorescence intensities. Reaction of GSH or a model peptide with HOCl produced the respective sulfinic (RSO2H) and sulfonic RSO3H acids, which would not be reduced to the parent thiols by DTT. However, parallel studies showed the effects of HOCl on mBBr-derived protein fluorescence to be more effectively reversed by DTT than were the effects of equal doses of GSSG. Protein aggregation was observed in the samples incubated with GSSG by the appearance and dose-dependent increase in intensity of bands retained at the top of the gels and decreases in intensities of smaller proteins. This apparent aggregation was not reversed by reduction with DTT, even with increasingly vigorous efforts to denature the proteins with SDS and with heating. The present results demonstrate that the effects of GSSG formation and PSH oxidation may not be as readily reversible as is often assumed and suggest that the formation of protein aggregates may be an understudied consequence and potential pathogenic factor in oxidant stresses.

Supported by GM44263 from NIH.