- Part 1:
White muscle disease (WMD), a degenerative muscle disease brought on by a deficiency of the essential trace nutrient selenium, is thought to be mediated by the protein Selenoprotein W (SeW). In its relationship with WMD, SeW, like many selenoproteins, binds to glutathione, serving some purpose in redox mechanisms in rats and in vivo. Recognizing that the protein may selectively bind glutathione and that it may be a glutathione-dependent in-vivo antioxidant, it has been proposed by Bauman (2004) that SeW has a mechanism involving reducing activity, much like that of thioredoxin reductase. This and the presence of selenocysteine in SeW suggest that there may be intramolecular disulfide/selenosulfide bonds being formed in the N-terminal region (this region contains Y-C-G-A-SeC or CXXSeC). Investigation into the possible presence of disulfide bonds in the non-native form of SeW, using elctrophoresis and MALDI-MS/MS, strongly suggest that there is a disulfide bond in the N-terminal region. A similar investigation into the presence of a selenosulfide bond in the native form of SeW, using samples isolated in the Bauman study, was inconclusive, due to the possible lack/low concentration of protein in the original samples (<10-100fmol), its possibly degraded state, or inappropriate procedure for analyzing the protein. It is still supposed that there is a selenosulfide bond in the N-terminal region of the native SeW, as in the non-native form, but until there is a sufficient amount of the protein to work with, this hypothesis cannot be tested.
High-energy radiation damage in biological macromolecules is responsible for diseases, aging, and impairment of vital biological functions. The radiation itself may be directly responsible for only part of the damage, though. Damage caused by intermediate species created within nanoscopic volumes along the ionization tracks can be far more significant because of the chemical reactions that follow. The intermediate species may consist of excited atoms and molecules, ions, radicals, and most importantly secondary electrons that are produced in very high abundance (>104 by a 1MeV projectile). Although much is known about secondary electrons and their interaction with DNA, little is known about the effect of such electrons on proteins even though interaction with secondary electrons resulting from irradiation is probable. In the case of caractacts, secondary electrons have been shown to cause a shuffling of disulfide linkages in lens proteins, which may undergo reduction by capture of low energy electrons then reoxidize forming non-native disulfides. A preliminary study was proposed, using Glutatione (GSH and GSSG, 100uM) as a model, which looked into the effects of secondary electrons on proteins with disulfide bonds, using varying levels (0-6.4kJ/m2) of UVB and UVC radiation, a DTNB assay, and HPLC-ESI-MS/MS. As the energy was increased step-wise (0-6.4kJ/m2), a 9.5±1.3% and 8.8±2.1% decrease in GSH activity was observed for 254nm and 365nm, respectively. This decrease could be from photo-oxidation. As the energy was increased step-wise from 0-32 MEDS (0-672 mJ/cm2), an increase of over 1uM was seen at 254nm. This increase could be a product of photolysis or photo reduction. To confirm assay findings, HPLC-ESI-MS/MS was attempted, but separation of the GSSG and GSH(-NEM) species could not be achieved and results were subsequently inconclusive.
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