title: Carbonyl-reducing enzymes as targets of a drug-immobilised affinity carrier.
abstract: Proteins, peptides and nucleic acids are commonly isolated and purified in almost all bioscience laboratories. Methods based on molecular recognition are currently the most powerful tool in separation processes due to their selectivity and recovery. The aim of this study was to prove the versatility and the ability of an affinity carrier containing the immobilised ligand oracin (previously developed by our workgroup) to selectively bind carbonyl-reducing enzymes. These enzymes play an important role in metabolic pathways of various endogenic compounds and xenobiotics. Many important drugs, such as doxorubicin, daunorubicin, haloperidol and the model anticancer drug oracin, are metabolised by carbonyl-reducing enzymes. The functionality of the presented carrier was demonstrated with pure recombinant enzymes (AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C3, AKR1C4, CBR1 and CBR3) as well as with two model biological samples (cell extract from genetically modified Escherichia coli and pre-purified human liver cytosol). Enzymes that show an affinity toward oracin were efficiently captured, gently eluted using 150 mM ammonium hydroxide and subsequently identified by MS. The method is highly selective and robust and may be applied to the purification and identification of various carbonyl-reducing enzymes from any biological sample.
authors.affiliation: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, Hradec Králové 500 05, Czech Republic. Electronic address: andrysr@faf.cuni.cz.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Andrýs
authors.affiliation: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, Hradec Králové 500 05, Czech Republic. Electronic address: zemanol9@faf.cuni.cz.
authors.forename: Lucie
authors.initials: L
authors.lastname: Zemanová
authors.affiliation: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, Hradec Králové 500 05, Czech Republic; Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, Třebešská 1575, Hradec Králové 500 01, Czech Republic. Electronic address: lenco@pmfhk.cz.
authors.forename: Juraj
authors.initials: J
authors.lastname: Lenčo
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentská 573, Pardubice 532 10, Czech Republic. Electronic address: zuzana.bilkova@upce.cz.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Bílková
authors.affiliation: Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, Hradec Králové 500 05, Czech Republic. Electronic address: wsol@faf.cuni.cz.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Wsól
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D064753:Ammonium Hydroxide / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004337:Drug Carriers / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D004983:Ethanolamines / Q000378:metabolism; D006801:Humans; D007546:Isoquinolines / Q000378:metabolism; D008024:Ligands; D008099:Liver / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*
chemical_list: D004337:Drug Carriers; D004983:Ethanolamines; D007546:Isoquinolines; D008024:Ligands; D004364:Pharmaceutical Preparations; C103439:oracine; D064753:Ammonium Hydroxide; D000429:Alcohol Oxidoreductases
keywords: Affinity chromatography; Carbonyl-reducing enzymes; Chemical proteomics; Magnetic microparticles; Oracin