Chemical Advances in Detecting Hydrogen Sulfide in Biological Systems
| dc.contributor.advisor | Johnson, Darren | |
| dc.contributor.author | Montoya, Leticia | |
| dc.date.accessioned | 2015-08-18T23:11:57Z | |
| dc.date.issued | 2015-08-18 | |
| dc.description.abstract | Hydrogen sulfide (H2S) is an essential biological signaling molecule for multiple regulatory pathways; however, H2S is also known to have toxic effects. Currently, there are few biocompatible methods available for studying its biological roles, and a major challenge in developing H2S detection methods is selectively separating the reactivity of H2S from that of other sulfhydryl-containing compounds. To address this challenge, we report on a colorimetric analytical method that is based on nitrobenzofurazan-derived electrophiles to facilitate H2S detection in biological/environmental applications, which provides one of the most sensitive colorimetric methods for H2S detection and quantification. We further evaluated the reactivity profiles of biological sulfhydryl-containing compounds with common electrophilic fluorescent thiol labeling reagents, toward cysteine and H2S. These studies highlight that the fluorescence from thiol-activated nitrobenzofurazan-based reagents is diminished in the presence of H2S. During the growth of the H2S field, monobromobimane (mBB) has emerged as a prominent method to detect and quantify sulfhydryl-containing compounds by fluorescence HPLC. We expand on this technique with the use of dibromobimane (dBB) for sulfide quantification. Reaction of dBB with sulfide is significantly faster than the reaction of sulfide with mBB, resulting in a highly fluorescent bimane thiother product, and affords a significantly better detection limit than mBB. Unfortunately, the use of dBB for quantification of H2S in biological samples results in extraction of sulfur from other sulfhydryl sources. We improved on the selectivity of H2S detection within biological samples by developing two bright fluorescent probes (HSN1/HSN2) that have a robust fluorescence turn-on, are selective for H2S, maintain low detection limits, and are applicable in live cells. Expanding on the biological applications of these sensing platforms, we report a method that utilizes HSN2 in combination with fusion proteins to detect endogenous H2S at subcellular resolution. Additionally, we demonstrate that H2S donors localize their sulfur release within different organelles of the cell. This methodology provides the first method for investigating chemical signaling of H2S within organelles of the cell and enables the utility of using one fluorophore for all simultaneous H2S studies within the same biological system. This dissertation includes both previously published and unpublished co-authored material. | en_US |
| dc.description.embargo | 10000-01-01 | |
| dc.identifier.uri | https://hdl.handle.net/1794/19328 | |
| dc.language.iso | en_US | |
| dc.publisher | University of Oregon | |
| dc.rights | All Rights Reserved. | |
| dc.subject | Biothiol | en_US |
| dc.subject | Fluorescence | en_US |
| dc.subject | Gaseotransmitter | en_US |
| dc.subject | Hydrogen Sulfide | en_US |
| dc.subject | Imaging | en_US |
| dc.subject | Subcellular | en_US |
| dc.title | Chemical Advances in Detecting Hydrogen Sulfide in Biological Systems | |
| dc.type | Electronic Thesis or Dissertation | |
| thesis.degree.discipline | Department of Chemistry and Biochemistry | |
| thesis.degree.grantor | University of Oregon | |
| thesis.degree.level | doctoral | |
| thesis.degree.name | Ph.D. |
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