title: Efficient isolation of carbonyl-reducing enzymes using affinity approach with anticancer drug oracin as a specific ligand.
abstract: Carbonyl-reducing enzymes are important in both metabolism of endogenous substances and biotransformation of xenobiotics. Because sufficient amounts of native enzymes must be obtained to study their roles in metabolism, an efficient purification strategy is very important. Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c] isoquinoline) is a prospective anticancer drug and one of the xenobiotic substrates for carbonyl-reducing enzymes. A new purification strategy based on molecular recognition of carbonyl-reducing enzymes with oracin as a ligand is reported here. The type of covalent bond, ligand molecules orientation, and their distance from the backbone of the solid matrix for good stearic accessibility were taken into account during the designing of the carrier. The carriers based on magnetically active microparticles were tested by recombinant enzymes AKR1C3 and CBR1. The SiMAG-COOH magnetic microparticles with N-alkylated oracin and BAPA as spacer arm provide required parameters: proper selectivity and specificity enabling to isolate the target enzyme in sufficient quantity, purity, and activity.
authors.affiliation: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Skarydová
authors.forename: Rudolf
authors.initials: R
authors.lastname: Andrýs
authors.forename: Lucie
authors.initials: L
authors.lastname: Holubová
authors.forename: Hana
authors.initials: H
authors.lastname: Stambergová
authors.forename: Jana
authors.initials: J
authors.lastname: Kňavová
authors.forename: Vladimír
authors.initials: V
authors.lastname: Wsól
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Bílková
mesh_terms: D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D000970:Antineoplastic Agents / Q000737:chemistry*; D001681:Biological Assay; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays / Q000379:methods*; D004798:Enzymes / Q000302:isolation & purification*; D004983:Ethanolamines / Q000737:chemistry*; D007546:Isoquinolines / Q000737:chemistry*; D008024:Ligands; D008280:Magnetics; D008863:Microspheres; D015394:Molecular Structure; D012545:Schiff Bases / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D004798:Enzymes; D004983:Ethanolamines; D007546:Isoquinolines; D008024:Ligands; D012545:Schiff Bases; C103439:oracine; D000429:Alcohol Oxidoreductases