The use of immobilized enzymes has steadily increased in recent years. Based upon the advantages that immobilized enzymes possess over soluble enzymes, numerous applications have emerged in medical and analytical fields. This work demonstrates the applicability of a liquid chromatographic system based upon coupled on-line immobilized enzyme reactors (IMERs) to organic synthesis, biochemistry and pharmacology. It is envisioned that the model system will grow into a modular process where synthetic chemists can add or subtract the enzymes necessary for their particular synthetic goal. The system allows for on-line chromatographic purification and structural identification of products and could greatly reduce time required to discover new synthetic pathways. In addition, the construction of a coupled enzyme system provides a number of approaches to basic research into synthetic and metabolic pathways as well as a rapid method for the discovery of new pharmaceutical substances. / A coupled system using extremely different enzymes with incompatible cofactors and reaction conditions has been constructed. The significance of the proposed project not only lies in the development of the liquid chromatographic on-line enzyme cascade but also in the biosynthetic pathway chosen for this study. The biosynthetic pathway involving dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase encompass the synthesis of the key transmitters, norepinephrine and epinephrine. The results demonstrate for the first time the immobilization of dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase. The IMERs are active and can be used in a liquid chromatographic format for qualitative and quantitative determinations. Studies with the IMER-HPLC systems have also shown that the activity of the immobilized enzymes reflects the non-immobilized enzymes. Thus, the IMER-HPLC system can be used to carry out standard Michaelis-Menten enzyme kinetic studies and to quantitatively determine enzyme kinetic constants, identify specific enzyme inhibitors, provide information regarding the mode of inhibition and the inhibitor constants (Ki). The immobilized enzyme reactors used independently or as a combination will provide a unique opportunity to explore the interrelationships between these enzymes, to investigate the source of catecholamine-related disorders and to design new drug entities for identified clinical syndromes.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37655 |
| Date | January 2001 |
| Creators | Markoglou, Nektaria. |
| Contributors | Wainer, Irving W. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001861905, proquestno: NQ78727, Theses scanned by UMI/ProQuest. |
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