It is proposed to investigate the biological chemistry of sulfur in the thiol and disulfide forms and selenium in the selenol and diselenide forms. The research will focus on chemical reactions which are central to the biological chemistry of sulfur and selenium, but which have not been quantitatively characterized because of a lack of experimental methods. Reactions will be characterized at the molecular level in intact erythrocytes, plasma and aqueous solution by NMR spectroscopy. Also, sensitive and selective methods will be developed for the determination of thiols, disulfides, selenols, diselenides and selenosulfides in biological fluids and erythrocytes by high performance liquid chromatography (HPLC) with electrochemical detection. The studies of sulfur and selenium compounds in intact erythrocytes will be designed to characterize both their transport across the erythrocyte membrane and their intracellular metabolism. These studies will take advantage of the unique capability of noninvasive 1H-NMR methods to provide information not only about membrane transport but also about the fate of the transported molecule. The HPLC methods will be used to determine metabolites formed at trace levels. The kinetics and equilibria of the reaction of thiols with disulfides and with diselenides will be characterized in aqueous solution and in plasma by 1H and 13C-NMR. Also, the reaction of selenite with thiol groups in biological molecules, which is thought to be a means of incorporation of selenium into living systems, will be studied by 1H, 13C and 77Se-NMR. The emphasis will be on characterizing the kinetics and the products of the reaction, and developing 77Se-NMR as a technique for probing the incorporation of selenium not only into small molecules but also proteins by this reaction. The proposed research will yield new and fundamental information about the transport of sulfur and selenium-containing molecules into erythrocytes, their metabolism in erythrocytes, and the intrinsic reducing strength of thiol and selenol groups in a variety of biological molecules. Also, information will be obtained about reactions which are models for the selenium-containing enzyme glutathione peroxidase. The HPLC methods to be developed will be a significant addition to the methods available for studying sulfur and selenium metabolism.
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