title: Recovery of biological active catechol-O-methyltransferase isoforms from Q-sepharose.
abstract: The development of new catechol-O-methyltransferase inhibitors has led to an improvement in the treatment of Parkinson's disease. However, despite the fact that the soluble isoform has been extensively investigated, few studies have been published concerning membrane isoform chromatographic recovery and bioactivity levels. In this work, chromatographic profiles of both catechol-O-methyltransferase isoforms were compared using quaternary amine as a ligand to evaluate its activity levels and recovery rates. Results show that both proteins required different conditions for adsorption; the soluble isoform adsorption was performed at low ionic strength, while the membrane isoform required increasing linear salt gradient. However, the application of 0.5% Triton X-100 promoted membrane isoform adsorption even at low ionic strength. Indeed, chromatographic conditions of both isoforms became similar when detergents were applied. The developed methods also appear to be highly effective in bioactivity recovery, presenting rates of 107% for soluble protein and 67 and 91% for membrane isoform without and with detergents, respectively. The chromatographic strategies with and without detergents resulted in a 4.3- and sevenfold purification, respectively, corresponding to specific activity values of 331 and 496 nmol/h/mg. Thus, the use of Q-sepharose as anion exchanger was effective in the recovery of both enzymes, which is a requirement for further kinetic and pharmacological trials.
authors.affiliation: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal.
authors.forename: F F
authors.initials: FF
authors.lastname: Correia
authors.forename: F M
authors.initials: FM
authors.lastname: Santos
authors.forename: A Q
authors.initials: AQ
authors.lastname: Pedro
authors.forename: M J
authors.initials: MJ
authors.lastname: Bonifácio
authors.forename: J A
authors.initials: JA
authors.lastname: Queiroz
authors.forename: L A
authors.initials: LA
authors.lastname: Passarinha
mesh_terms: D000327:Adsorption; D000837:Anion Exchange Resins / Q000737:chemistry; D002394:Catechol O-Methyltransferase / Q000737:chemistry / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D012685:Sepharose / Q000737:chemistry*
chemical_list: D000837:Anion Exchange Resins; D007527:Isoenzymes; D012685:Sepharose; D002394:Catechol O-Methyltransferase
keywords: Anion exchange chromatography; Catechol-O-methyltransferase; Membrane proteins; Parkinson's disease