Jiang, Lingxi2018-12-172018-12-172018http://udspace.udel.edu/handle/19716/23995The extracellular SH/SS redox status has been shown to play critical roles in a range of mammalian cell behaviors, including migration, proliferation, cancer invasion, and virus fusion. Quiescin-sulfhydryl oxidase (QSOX), a facile disulfide-generating enzyme discovered in our laboratory, is believed to contribute to extracellular SH/SS status. Although the enzymological properties of QSOX have been studied extensively in our laboratory, its contribution to disulfide bond formation in biological contexts is still cryptic. This dissertation includes two projects that impact extracellular redox state regulation. In the first part of this work, a simple and sensitive fluorescence-based microplate assay for QSOX activity is described. This assay couples hydrogen peroxide formation generated by sulfhydryl oxidases to the generation of the strong red fluorescence formed during HRP-catalyzed oxidation of Amplex UltraRed and is suitable for testing QSOX activity in small samples of serum, plasma, or other biological fluids. This work shows that murine, bovine, and human sera contain significant levels of sulfhydryl oxidase activity. We also found similar levels of enzymatic activity between fetal and adult bovine sera in contract to a prior report. A three-step purification protocol using adult bovine serum showed that this activity reflects circulating, soluble QSOX1. Peptide digests and mass spectrometric analysis confirmed that this disulfide-generating activity was indeed due to QSOX1. The presence of a facile oxidase for a wide range of thiol-containing peptides and proteins in mammalian plasma suggests a new dimension to the study of thiol/disulfide redox biochemistry in blood. In the second project, novel ratiometric fluorescence imaging methods for surface thiols and disulfides and their applications to studying a range of cellular phenomena were described. Membrane-impermeant fluorescent maleimide reagents were used throughout, and these new methods can visualize and quantitatively assess the status of surface proteins on normal and cancer cells. In addition, a dramatic effect of very low concentrations of polystyrene sulfonate (PSS) on cell surface SH/SS redox status was investigated. This effect is thiol specific, general to different cell types, and reversed, in part, by exogenous QSOX. PSS is also found to increase the endocytosis of exofacial thiols and proteins. Finally, we showed that the new ratiometric methods can be extended to multicellular organisms and biomaterials. These procedures are amenable for future super-resolution studies.Pure sciencesBiological sciencesHealth and environmental sciencesDisulfide bond formationFluorescence-based microplate assaySurface proteinsThesis1079055730https://doi.org/10.58088/ay9m-0t842018-10-17en