title: Analysis of hSCOMT adsorption in bioaffinity chromatography with immobilized amino acids: the influence of pH and ionic strength.
abstract: In the last years, chromatographic supports with amino acids as immobilized ligands (AAILs) were been used successfully for isolation of several biomolecules, such as proteins. In this context and based on specific properties of human soluble cathecol-O-methyltransferase (hSCOMT), we screened and analyzed the effect of experimental conditions, such as pH and ionic strength manipulation for hSCOMT adsorption, over six different AAIL commercial supports. Typically, the proteins adsorption on AAIL chromatographic supports is around their pI. While hSCOMT isoelectric point is around 5.5, this parameter leads us to design new adsorption strategies with several acid buffers for the chromatographic process. In terms of the ionic strength manipulation strategy, the results suggest that the AAILs-hSCOMT interaction is strongly affected by the intrinsic hSCOMT hydrophobic domains. On the other hand, the interaction mechanism of hSCOMT on amino acid resins appears to be highly dependent on the binding pH. Consequently the retention mechanism of the target enzyme on the AAILs can be as either in typical hydrophobic or ionic chromatographic supports, so long as selecting various mobile phases and separation conditions. In spite of these mixed-mode interactions and operation strategies, the elution of interferent's proteins from recombinant host can be achieved only with suitable adjusts in pH mobile phase set point. This lead to a new approach in biochromatographic COMT retention, while possess a higher specificity than other chromatographic methods reported in literature.
authors.affiliation: CICS-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal.
authors.forename: S R
authors.initials: SR
authors.lastname: Costa
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; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D002394:Catechol O-Methyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000596:Amino Acids; D055767:Immobilized Proteins; D011994:Recombinant Proteins; D002394:Catechol O-Methyltransferase