Date of Award

2003

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Mutus, Bulent,

Keywords

Chemistry, Biochemistry.

Rights

CC BY-NC-ND 4.0

Abstract

Nitric oxide is involved in many physiological processes including vascular control, immune responses and neurotransmission under physiological conditions. Many functions of NO are triggered by the formation of its metabolite, S-nitrosothiol. S-nitrosothiols play an important role in the delivery, storage and transport of NO. In addition, they regulate the activity of a variety of proteins and enzymes. The first part of my study is focused on studying the regulatory role of S-nitrosothiol. In this study, S-nitrosothiols including S-nitrosoglutathione (GSNO) have been demonstrated to regulate the activity of a key protein in plasma, fibrinogen, without chemically modifying it. GSNO and a few other RSNO derivatives have demonstrated the ability to inhibit thrombin catalyzed fibrinogen polymerization via their effects on fibrinogen, whereas the activity of thrombin itself remained intact upon incubation with GSNO. The percentage of inhibition obtained ranged from 43% to 68%. Upon incubation with GSNO, the alpha-helix content of fibrinogen increased by 15%. The GSNO fibrinogen interaction was allosteric and reversible with an estimated dissociation constant of 3-10 muM. Fibrinogen has been demonstrated to contain 2 binding sites for GSNO. The second part of my study deals with the chemical and cellular aspects of S-nitrosothiols. I formed a coloured nitrite adduct of sinapinic acid (SA) which has shown the ability to S-nitrosate thiol-containing amino acids and proteins. In addition, this nitrite adduct has demonstrated the potential for spectrophotometric detection of NO derived species, NO+ or peroxynitrite in vitro or under physiological conditions. In addition, I synthesized an S-nitroso derivative of 1-octadecane thiol, S-nitrosooctadecane (SNOD). We also designed SNOD-BSA nanoparticles, which were capable of delivering large amounts of SNOD to human fibroblasts. In preliminary studies, the illumination of SNOD-BSA loaded fibroblasts induced apoptosis in 58% of the fibroblasts. The third part of my study deals with the molecular aspects of S-nitrosothiols. Under physiological conditions, the reaction between NO and O2 •- produces peroxynitrite. I have shown previously that upon exposure to light, an air-saturated GSNO solution can also give rise to peroxynitrite. Peroxynitrite causes inactivation of many proteins and enzymes by nitrating their tyrosine residues. We created three different tyrosine mutants of rat calmodulin namely, CaM Y99A CaM Y138A, and CaM Y99AY138A for assessing the roles of its two tyrosine residues. Mutations of the tyrosine residues apparently affected calmodulin's stability and its Ca2+ binding ability. S-nitrosothiols are also capable of causing DNA damage. For the purpose of decontaminating platelet-rich plasma, we synthesized two fluorophore labelled S-nitrosthiols namely N-dansyl-S-nitrosohomocysteine (Dns-HCysNO) and N-dansyl-S-nitrosoglutathione (Dns-GSNO), which could mediate the cleavage of DNA upon exposure to light. Dns-HcysNO solutions degraded R773 plasmid DNA completely upon exposure to light and the extent of degradation was a function of exposure time. In contrast, only a high concentration of Dns-HCysNO degraded plasmid DNA partially without exposure to light. Thus, Dns-HCysNO could be potentially utilized for light-induced DNA cleavage in order to decontaminate platelet rich plasma.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2003 .A44. Source: Dissertation Abstracts International, Volume: 64-08, Section: B, page: 3800. Adviser: Bulent Mutus. Thesis (Ph.D.)--University of Windsor (Canada), 2003.

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