Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.6/3923
Título: Screening of Bioaffinity ligands for human soluble COMT purification
Autor: Costa, Sara Rute Afonso
Orientador: Passarinha, Luís António Paulino
Queiroz, João António de Sampaio Rodrigues
Palavras-chave: Catecol-O-Metiltransferase (COMT)
Cromatografia de interacção hidrofobica (HIC)
Cromatografia de pseudo-bioafinidade
Purificação
Data de Defesa: 2010
Resumo: Catechol-O-methyltransferase (COMT), play an important role in the metabolism of catecholamines, catecholestrogens and catechol drugs and consequently has a closer relationship with several mental disorders. As a result, while the development of pharmaceutical Human Soluble COMT (hSCOMT) trials for a rational drug design depends on the availability of high purified samples, more suitable purification strategies must be developed and emerged in order to fulfil the requirements of pharmaceutical industry. In this context, the global aim of this work was the improvement of recovery yields and activity protein results in a hSCOMT purification process. Therefore, the work was being oriented according to three intermediate goals. Two first’s concerning Hidrophobic Interaction Chromatography; (1) Octyl-sepharose support in order to analyse new fractionation methods by testing destabilizing elution conditions with the incorporation of L-arginine in the mobile phase; (2) the application of a dual salt system in Epoxy-sepharose resin; (3) the pseudo-bioaffinity chromatography using six commercial resins with immobilized amino acids, examining the ligands performance, in order to understand the selectivity’s achieved based on non-specific desorption profiles supported on pH and ionic strength manipulation. In general for Epoxy-sepharose support, the concentrations of dual salt system required to allow hSCOMT retention was above 0,7 M NH2SO4/0,15 M Na3C6H5O7. Indeed, this strategy is needless, considering previous purification strategies described that allow satisfatory protein selectivity and purification factor with less salt concentration; and consequently with a reduction in hSCOMT denaturation effects. The L-arginine is highly effective in improving the performance of various chromatographic columns. However, is this study preliminary stability assays showed that this amino acid decreases hSCOMT specific activity; showing that this strategy do not comprise hSCOMT purification strategy requests. This is the first report using amino acids as immobilized ligands (AAIL’s) as pseudo-bioaffinity supports in hSCOMT isolation. Based on non-specific desorption profiles the results show that; (1) the high concentrations necessary to promote hSCOMT retention, decreasing product activity recovery; (2) for pH approach, the requirement of acidic pH’s to allow hSCOMT retention leads to a molecular weight alteration and consequently loss of enzymatic activity. The comparison between these two strategies, reveal that the hSCOMT molecular weight discrepancy are not AAIL dependent, but due to binding conditions. In conclusion, the comparison of these three approaches in this dissertation demonstrated the complexity of hSCOMT purification processes. In spite of, structural studies are need to understand the hSCOMT-AAIL binding mechanism, these supports have furthermost advantages over the earlier methods published due of its simplicity and efficiency for hSCOMT purification.
URI: http://hdl.handle.net/10400.6/3923
Designação: Mestrado em Bioquímica
Aparece nas colecções:FC - DQ | Dissertações de Mestrado e Teses de Doutoramento

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