Date of Award

9-27-2018

Degree Type

Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

First Advisor

Mutus, Bulent

Keywords

GSNO, GSNOR, HDX-MS, mass spectrometry, nano particles, nitritc oxide

Rights

CC-BY-NC-ND

Abstract

Nitric oxide (NO) is a vital gasotransmitter, involved in a plethora of signaling pathways. With bioavailable NO stored as S-nitrosoglutathione (GSNO), its enzymatic degradation by S-nitrosoglutathione reductase (GSNOR) has a large impact on cellular SNO levels. This thesis consists of two components: chapter one explores the production of NO generating particles, that use chitosan as a matrix for a copper catalyst. Chapter two reports the discovery into a new regulatory domain on the GSNOR enzyme. In chapter one, we have developed a novel technology capable of generating NO, in the form of NO-releasing copper-chitosan particles (Cu-chito). These particles were successfully tested in their release of authentic NO: the maximum rate of production was 1.40 nmol/min/g. The Cu-chito particle treatments were analysed using scratch assays. In comparison to control groups: Cu-chito particles, which provide bioactive NO, displayed 130% of cells in the wound, while treatments with supplemented glucose displayed 152%. This supports the hypothesis that glucose aids to regenerate active copper in the catalytic cycle of NO production. Matrix metalloproteinases (MMP-2 and -9) were chosen as an intracellular NO target. The activity MMP was investigated: displayed an increase of 60% after Cu-chito treatments. The NO treatments provide a lasting effect on a cellular level. Presented in chapter two is the discovery of a new regulatory domain on GSNOR: an allosteric binding domain. A sigmoidal deviation in GSNOR kinetics indicated positive cooperativity for GSNO binding. Molecular docking (MD) simulations indicates the location a putative allosteric site at the amino acid residues Gly321, Lys323, Asn185, and Lys188. To further these studies hydrogen / deuterium exchange (HDX) mass spectrometry (MS) experiments were performed. With a two second HDX reaction, the residues Gly321, Lys323, and Lys188 displayed a decrease in deuterium uptake of 1.4%, 1.4%, and 0.4%, after the incorporation of GSNO. These results strongly support the existence of a secondary binding domain of substrate GSNO that contributes to its mechanism of action.

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