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In this work, the chemical preparation and characterization of an isotopically enriched superoxide dismutase (SOD) is described. Its evaluation as a standard in species-specific isotope dilution analysis by HPLC coupled to ICP-MS is carefully evaluated. The proposed method involved the removal of the enzyme's metal co-factors under various conditions and their replacement with isotopically enriched ⁶⁵Cu and ⁶⁸Zn. SEC-ICP-MS showed that the prepared enriched enzymes had a different metal isotopic abundance compared to the wild-type enzyme. Isotopically enriched and wild-type SOD showed the same migration pattern in 1D-PAGE. An enzyme activity assay provided evidence that incorporated ⁶⁵Cu was bound to the correct SOD-binding motif, since the measured activity correlated directly with the amount of Cu present in the prepared enzyme. The addition of free Cu and Zn or a metal chelator did not result in any exchange or loss of metals from the enzyme at neutral pH. Striking experiments were undertaken to evaluate the use of isotopically enriched SOD in SS-IDMS. The chemical preparation study on SOD was further extended to prepare various other isotopically enriched metalloproteins, including carbonic anhydrase, ceruloplasmin, transferring and haemoglobin. Various enrichment procedures were conducted and their performances then evaluated, using SEC-ICP-MS and protein assays. A procedure for the quantification of SOD in tissue samples using an isotopically enriched SOD spike in combination with 2-dimensional HPLC and SS-IDMS was developed and assessed. The feasibility of employing isotopically enriched protein spikes for the speciation of metalloproteins by utilising gel electrophoresis and LA-ICP-MS was also investigated. Furthermore, the change of the iron speciation of the meat-containing protein myoglobin after various treatments was examined using a combination of SEC-ICP-MS and ESI-MS.

Iron is an essential micronutrient that is required by some microorganisms in relatively large quantities. This is problematic for those inhabiting marine environments, where iron is highly insoluble and the dissolved fraction is predominantly strongly bound to organic compounds. Due to low supply and high demand, iron limits primary productivity in many oceanic waters, and may also limit growth of organisms in coastal waters under some circumstances. Recent incidents of explosive growth (???blooms???) of the noxious filamentous cyanobacterium Lyngbya majuscula in the coastal marine waters of Moreton Bay, Queensland, have prompted speculation that terrestrial human activities have increased iron availability to the organism, thus overcoming previous limitations on growth imposed by scarcity of the nutrient. This thesis describes work investigating the chemical form of iron in coastal waters under various environmental conditions and the way in which this influences its availability to L. majuscula. Chemical speciation of iron was investigated as a function of terrestrial-derived inputs of natural organic matter (NOM) of variable origin and sunlight in coastal marine waters, employing chemiluminescence-based and spectrophotometric techniques with high sensitivity and temporal resolution. These techniques allowed determination of iron and other chemical parameters at naturally occurring (typically nanomolar) concentrations. The mechanism of iron acquisition by L. majuscula was also investigated using a radioisotope-labelling labelling technique in addition to the other techniques described. Results indicated that iron speciation can be described by five classes: inorganic dissolved and organically complexed dissolved iron in both ferrous (reduced) and ferric (oxidised) forms, and precipitated inorganic iron. Simulation of laboratory results by numerical kinetic modelling of the processes investigated indicated that while the thermodynamic impetus is strongly towards precipitated iron, iron complexation by NOM and its reduction by sunlight-mediated processes and/or L. majuscula results in meta-stable dissolved species that are more readily available to L. majuscula. Superoxide is a critical intermediate in iron reduction by both sunlight and L. majuscula. Thus L. majuscula is capable of altering iron speciation to increase its availability, however uptake is also strongly dependent on environmental conditions and may be enhanced by increased inputs of iron, NOM and sunlight into coastal waters.

Superoxide, a reduced form of dioxygen is produced in natural waters from abiotic thermal processes such as iron(II) oxygenation photochemically, and biologically in natural waters. Superoxide is highly redox-active due to the presence of an unpaired electron and plays an important role in the geochemical cycling of trace metals and degradation of organic pollutants in natural waters. It also has a significant effect on the physiology of aquatic microorganisms and has been implicated in the growth of toxic microalga in coastal areas of Japan, Australia and Canada. The generation of superoxide by both biotic and photochemical pathways is described in this thesis and attention given to selected reactions of superoxide in simulated natural waters. Particular attention in this work has been given to the interaction of superoxide and iron and the impact of this interaction on iron availability to Chattonella marina, a red-tide phytoplankton frequently associated with fish-mortalities in Australia and Japan. Superoxide production from both phototchemical and biological sources is measured using a highly sensitive chemiluminescence technique and the effect of superoxide production on iron transformation under various environmental conditions is investigated by employing spectrophotometric techniques with high sensitivity and temporal resolution. The intermediacy of superoxide in iron acquisition mechanism by C. marina is also investigated using a radioisotope labeling technique. Our experimental results show that superoxide in natural waters is produced via reduction of dioxygen. The reduction of dioxygen takes place by transfer of electrons by the photo-excited quinone moieties present in natural organic matter or by reductases located on the outer cell membrane of organisms' surface. The experimental results suggest that the fate of iron in marine waters is closely related to the superoxidedioxygen redox couple. Superoxide can reduce a wide range of organically complexed iron(III) species to the more soluble iron(II) redox state, thus affecting its bioavailability. A simple kinetic model for redox-cycling of iron in the presence of superoxide is developed. In addition, by coupling the model for redox-cycling of iron with the forms of iron acquired by C. marina, a generalized mathematical model for iron acquisition is presented which satisfactorily describes all results obtained.

"March 2002" / Includes bibliographical references. / xii, 178 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the application of physico-chemical phenomena to aspects of environmental analysis. Measures the superoxide radical in natural waters in order to establish the role of the radical in natural water chemistry. Also investigates the electrokinetic charging behaviour associated with liquid microjets in order to understand the unusual charging behaviour reported in the literature with the ultimate goal of determining the viability of using the charging mechanism for in vacuo ion generation. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 2002

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Taiwan. Copper-zinc superoxide dismutase (SOD1) is widely distributed and comprises 90% of the total superoxide dismutase (SOD), which catalyzes the conversion of superoxide to hydrogen peroxide. Reduced expression of antioxidant enzymes, particularly SOD1, has been identified in human hepatoma specimens and cell lines. However, it remains unclear how SOD1 expression affected the tumorigenic processes of hepatoma cells. Expression analysis of an array of human HCC cell lines revealed that SOD1 protein levels were down regulated in poorly differentiated SK-Hep-1 cells. Adenovirus-mediated SOD1 expression increased the SOD1 protein level by 30-40% of control. In addition, SOD1 gene transfer decreased the cellular O2 ¡V level yet increased the H2O2 production. SOD1 overexpression significantly reduced the proliferation, motility, and anchorage-independent growth of SK-Hep-1 cells, but had no effect on the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9. SOD1 restoration inhibited the proliferation of SK-Hep-1 cells through induction of cell cycle arrest, which was associated with decreased expression of cyclin A, cyclin D1, cdk1, cdk4 and upregulation of p21Cip1 and p27kip1. Besides, SOD1 overexpression also inhibited the nuclear factor £e B (NF-£eB) activities, thereby attenuating the proliferation and migration of SK-Hep-1 cells. In conclusion, SOD1 restoration attenuated the tumorigenicity of hepatoma cells.

Amyotrophic lateral sclerosis (ALS) is a devastating, progressive, and fatal neurodegenerative disease. Despite the fact that ALS is the most common motor neuron disease in adulthood, there is no effective treatment for the disease. Although most ALS cases (90-95%) are sporadic (sALS), the remaining cases (5-10%) are dominantly inherited and referred to as familial ALS (fALS). Because sALS and fALS show indistinguishable disease symptoms, a common disease mechanism has been proposed. After the discovery of the link between fALS and mutants of cytosolic Cu/Zn superoxide dismutase (SOD1), over 140 mutations in SOD1 have been identified to account for ~20% of fALS. The location of these mutants are scattered throughout the primary and tertiary structures of the protein. It is widely accepted that fALS-linked mutations in SOD1 result in a gain of toxic function to cause the disease, rather than a loss of physiological function, although the nature of the toxic mechanism remains unclear. SOD1 is a -rich, homodimeric metalloenzyme that catalyzes the dismutation of superoxide radicals to O2 and H2O2. The protein is ubiquitously expressed and the mature form of SOD1 (holo SOD1) contains one catalytic Cu ion, one structural Zn ion, one intra-molecular disulfide bond (between C57 and C146) and two free Cys residues (C6 and C111) per 153 residue subunit. Analogous to many different human diseases in which protein aggregation is a hallmark, aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in the pathogenesis of ALS. This thesis reports the first observation of aggregation of the most abundant form of SOD1 in vivo, the native, metallated (holo) dimer, under physiologically relevant conditions (37 °C and pH 7.8). The medical relevance of aggregates is demonstrated by structural and tinctorial analyses as well as the novel observation of binding of an anti-SOD1 antibody that specifically recognizes pathological aggregates in ALS. Additionally, ALS-associated SOD1 mutations promote aggregation but are not required, supporting a common mechanism in familial and sporadic ALS. The aggregation is characterized by a lag phase, which is diminished by self- and cross-seeding where heterogeneous nucleation is the underlying mechanism. Moreover, multiple pathways of aggregation are elucidated including dimer dissociation and metal loss. It is shown that if holo SOD1 loses more Zn ions than Cu ions, the aggregation profiles have shorter duration and lower final intensity, whereas when holo SOD1 loses more Cu ions than Zn ions, the aggregation profiles have longer duration and higher intensity. Taken together, the data in this thesis establish a valuable system for understanding SOD1 aggregation and toxicity mechanisms which can be used for developing therapeutic strategies targeting protein aggregation.

Thesis (M.Med.Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 79-86).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 164-173).

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