A PREVIEW TO VITAMIN D BIOSENSING: ELECTROCHEMICAL EVALUATION OF CYTOCHROME P450 27B1
Steve Y. Rhieu1,2 and G. Tayhas R. Palmore2
1Biochemical Sciences Division, National Institute of Standards and Technology, Gaithersburg MD 20899 2Division of Biology and Medicine, Brown University, Providence RI 02912
Recent research on the electrochemistry of cytochrome P450s (CYPs) has focused on elucidating their electron transport pathways and their development for use in biosensors and bioreactors. However, heterogeneous electron transfer is difficult to achieve in many oxidoreductases, especially with CYPs because membrane-bound enzymes exhibit a general instability when solubilized and the heme active-site is often buried within the protein. Much effort has been made to overcome the aforementioned issues. To date, coating an electrode surface with a surfactant film has been a successful method to facilitate direct electron transfer to heme-containing proteins such as myoglobin. The stereoselective hydroxylation of 25-hydroxyvitamin D3 (25(OH)D3) is catalyzed by CYP27B1. This reaction results in the formation of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the biologically active form of vitamin D3. The CYP27B1-catalyzed 1α-hydroxylation reaction required the uptake of two electrons from NADPH via an electron transport chain consisting of two proteins: a FAD-containing adrenodoxin reductase and an iron-sulfur protein, adrenodoxin. Recently, mouse CYP27B1 has been overexpressed in Escherichia coli, purified, and biochemically characterized. As such, the availability of recombinant CYP27B1 makes possible its electrochemical characterization. Here we report direct electrochemistry of recombinant mouse CYP27B1 immobilized on an edge-plane pyrolytic graphite electrode coated with a thin didodecyldimethylammonium bromide film. The electrochemical characterization of CYP27B1 presented in this work represents a first step toward measuring 25(OH)D3 electrochemically.