NADH and NADPH are ubiquitous biological reducing agents essential for both respiration and biosynthesis. The discovery that increased pentose-phosphate pathway activity in cervical cancer cells leads to increased levels of NAD(P)H, emphasises the need for a sensitive detection system as an indication of cellular viability and vitality. The remit of this project was to design and synthesise a novel molecular sensor system whose emissive properties are "switched on" upon reduction by NAD(P)H. Research using the reducible, non-fluorescent dye, resazurin, has shown that, in the presence of a non-enzymic electron transfer agent phenazinium methosulphate (PMS)-NADH can effect reduction to the highly fluorescent dye resorufin. Mechanistic studies have shown that the reduction proceeds via a two-electron hydride transfer to the heterocyclic mediator, followed by a one electron transfer to the dye and disproportionation to furnish the final fluorescent product. It has been shown that direct reduction by NADH does not occur and that the reaction depends upon there being an electron transfer agent present. A new type of reagent for the detection of NAD(P)H has been synthesised, comprising a reducible heterocycle and a masked fluorophore. It has been shown that reduction of the precursor conjugate by NADH results in the release of a detectable fluorescent moiety methylumbelliferone. The synthesis of an analogous conjugate probe containing a known hindered dioxetane moiety is described. Prepared using a previously unreported route, the key vinyl ether intermediate is generated via a Wadsworth-Emmons reductive coupling of an alkoxy phosphonate to 2-adamantanone. Reduction by NADH and subsequent cleavage of a conjugate ether link generates an electron rich phenolate substituted dioxetane which is metastable, resulting in emission from the generated excited product. Work towards a dioxetane containing functionalised alkyl group for conjugation to a fluorophore is also outlined.

No description available.

Pyridine nucleotide transhydrogenase catalyzes the reversible transfer of hydride ion equivalents between NADP(H) and NAD(H). In this study, the activity of the enzyme was measured by following the rate of reduction of an analogue of NAD⁺ , 3-acetylpyridxne-NAD⁺ (APNAD⁺ ) by NADPH. The enzyme was solubilized by detergents such as lysolecithin, sodium cholate (in the presence of ammonium sulphate) or Triton X-100. The molecular size of the solubilized enzyme was examined using sucrose density gradient centrifugation in the presence of Brij 58. These detergents gave soluble fragments of different sizes. That solubilized by Triton X-100 or sodium cholate (in the presence of ammonium sulphate) existed as large aggregates with sedimentation coefficients of 24.5 to 25.4S, whereas that obtained with lysolecithin consisted mainly of a species with a sedimentation coefficient of 7.3 to 16.5S. The fragment resulting from the solubilization with Triton X-100 could be cleaved into a smaller species (8.4S) by lysolecithin. Analysis by chromatography on Sepharose 6B of the enzyme preparation solubilized by sodium cholate (in the presence of ammonium sulphate), revealed the presence of other constituents of the membrane, such as succinate dehydrogenase, ATPase and cytochrome b₁. The molecular weight of the aggregate was estimated to be between 0.25 x 10⁶ and 4 x 10⁶. The enzyme in this preparation could not be further disaggregated by Tween 80, Brij 3 5 or Triton X-100. Chromatography of this preparation on DEAE-Sepharose CL-6B yielded a maximum purification of 37 to 68-fold over that of the membrane particle suspension. The specific activity of the enzyme was 8.8 to 15.7 umol per min per mg protein. Analysis of the partially purified enzyme on poly-acrylamide gels in the presence of sodium dodecyl sulphate revealed enrichment of several major polypeptide bands of molecular weights 90 000, 57 000, 50 000 and 40 000, coinciding with the transhydrogenase activity. The partially purified enzyme could be activated by detergents of the Tween or Brij series and by lysolecithin, palmitic acid and phospholipid extracts from E. coli. Measurements of the steady-state kinetics of the membrane-bound enzyme gave values of 45.6 and 106.7 uM for the substrates APNAD+ and NADPH, and dissociation constants of 3.6 and 16.2 uM, respectively. Lineweaver-Burk plots for each substrate at different fixed concentrations of the other substrate revealed a unique pattern of lines that is characteristic of rapid equilibrium random bireactant mechanisms with two dead-end products. In this type of mechanism each substrate is able to interact at the binding site of the other substrate to cause inhibition of enzyme activity. This mechanism was confirmed by kinetic studies using the alternate substrates deamino-NADPH and NAD⁺ , as well as by product inhibitxon studies. The adenine nucleotides 5’-AMP and ADP were competitive inhibitors of the APNAD+-binding site, while 2'-AMP was a competitive inhibitor of the NADPH-binding site on the enzyme. Studies on the active site using 2,3-butanedione or phenyl glyoxal revealed the presence of one modifiable arginyl residue per active site on the enzyme. Protection against modification by 2,3-butanedione was afforded by 2'-AMP, 5'-AMP, NAD+ and NADP+. Inhibition by 2,3-butanedione was enhanced in the presence of low concentrations of NADH or NADPH suggesting that binding of the reduced pyridine nucleotides, possibly at an allosteric site, causes a conformational change in the enzyme. Enhancement of in-activation of the enzyme by TPCK-trypsin was also observed in the presence of reduced pyridine nucleotides. NAD(P)H was oxidized by 2,3-butanedione in the presence of light. The rate of photooxidation was greatest at pH 7 and when the wavelength of incident light was 410 nm. This indicates that absorption of light by the diketone was necessary for the occurrence of the photooxidation reaction. The stochiometry of the reaction between NADH and 2,3-butanedione was 1:1. The possible nature of the reaction product is discussed in the thesis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 197 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 188-197). Also available online.