Electrochemistry of cytochrome P450 enzyme on nanoparticle-containing membrane-coated electrode and its applications for drug sensing

In the current study, we describe an improved system to study the two-electron delivery reaction pathway of cytochrome P450, family 2, subfamily B, polypeptide 6 (CYP2B6) in vitro. In particular, a biocompatible film containing colloidal gold nanoparticles and chitosan was used to encapsulate CYP2B6 on an electrode. The electrocatalytic behaviors of CYP2B6 toward common drugs in the absence of NADHP–cytochrome P450 reductase as electron donor were studied. In an anaerobic solution, direct and reversible electron transfer between the electroactive heme center of CYP2B6 and the electrode was observed with a formal potential of –0.454 ± 0.006 V at pH 7.4. In an air-saturated solution, an increase in the bioelectrocatalytic reduction current was observed after drug addition. The bioelectrocatalytic products were analyzed using high-performance liquid chromatography (HPLC) and electrospray ionization–mass spectrometry (ESI–MS). Both results confirmed that C-hydroxylation and heteroatom release were the main pathways for CYP2B6-mediated drug oxidation, similar to what occurred in vivo. The use of immobilized proteins in nanoparticle-containing films in drug biosensing was also demonstrated.