Specificity of cytochrome P450s in thin films

Date of Completion

January 2003


Chemistry, Biochemistry




Human liver cytochrome P450s (CYP) is a class of enzymes known to metabolize a wide variety of compounds. To ascertain toxicological effects of pharmaceutical new drug entities or to study drug-drug interactions, CYP activity assays are conducted. Long-term enzyme stability, the inability to reuse enzyme for multiple experiments, and the use of expensive biological cofactors are concerns when conducting these assays. Immobilization of enzyme increases its stability and the potential for its reusability. ^ The aim of this dissertation is to determine substrate specificities of immobilized CYPs, which is vital for assessing possible biosensor applications towards toxicity screening, environmental detoxification, and comparative turnover studies. Cytochromes P450 1A2 and P450cam were studied with styrene, cis-β-methylstyrene, and phenacetin as substrates. Turnover numbers were calculated for each substrate with enzyme either in solution or immobilized on films. The comparison of both solution and film TN's, while keeping all conditions the same, help determine if the specificities of enzymes are retained after immobilization. ^ Styrene is a compound whose CYP metabolite, styrene oxide, is known to form DNA adducts. Phenacetin is an activity marker compound for CYP 1A2. Its CYP metabolite, acetaminophen, is a common pain medicine. TN's of CYP 1A2 and cam for styrene and phenacetin are discussed. ^ Cis-β-methyl styrene was used to investigate the stereo- and enantioselectivities of immobilized cytochrome P450cam and 1A2. Metabolites formed were cis- and trans-β-methylstyrene oxides. Stereochemistry and enantioselectivity results are presented. ^