Carbonyl-reducing enzymes as targets of a drug-immobilised affinity carrier

• Separation protocol using affinity carrier with immobilized oracin was improved.
• Multiple carbonyl-reducing enzymes were successfully captured by affinity carrier.
• Enzymes with no affinity to oracin were not captured.
• Affinity carrier successfully isolated enzymes from complex biological samples.
• Isolated enzymes were obtained in native active form.

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.