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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:499324 |
| Date | January 2009 |
| Creators | Deitrich, Christian L. |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=25802 |
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