Cytochrome P450 (CYP) enzymes are a well-known family of drug-metabolizing enzymes to catalyze mono-oxygenation reactions of an enormous number and variety of substrates. It has been previously reported that they play roles in generation of reactive oxygen species (ROS), such as superoxide anion (O2-·), hydrogen peroxide(H2O2) and hydroxyl radical(·OH) during drug metabolism. If so, what is the concentration range of the released ROS? And how can we detect them?
Quantitative measurement of hydrogen peroxide can be done by chemical analysis. You can find numerous methods described in the literature. Direct measurements of radicals such as superoxide or hydroxyl are challenging and would require specialized ESR equipment. However, you can monitor chemical reactions resulting from hydroxyl radicals using appropriate substrate, and quantitate the effects of these radicals by measuring the product of the reaction. For hydroxyl radicals a very good substrates are compounds containing aromatic ring, and are readily amenable to hydroxylation. For instance, coumarin is very good substrate for monitoring enzymatic hydroxylation reaction of P-450. Here is a typical reaction for aromatic ring hydroxylation assay developed in our lab using P-450 isolated from the rat liver:
Reaction can be carried out in phosphate buffer. Substrate concentration: P-450; 2mM, coumarin; 0.0625mM up to 2mM, NADPH: 2mM, Incubation time; 10 minutes, Termination; Acetonitrile and extraction with Chlorophorm.
The product of the reaction is 7-hydroxy coumarin which can be measured using HPLC. Analysis; Column; PRP-1 - 10mm, Mobile Phase; 70% Acetonitrile, Flow; 1.5ml/min, Detection; fluorescence @ Ex 325nm; Em 445nm. Detection limit in pM range.
Hi Bin Guo,
The detection of hydrogen peroxide produced by either the P450 or the NADPH-P450 recutase is uneasy because of the very high unstability of hydrogen peoxide. P450 enzymes may produce H2O2 through uncoupling of their catalytic cycle. P450 reductase may produce H2O2 in some cases, but most H2O2 produced in P450 functioning is due to the P450 not to the reductase.
The measurement of H2O2 produced is based on the homovanillic acid assay and has been described in our lab almost twenty years ago. Please look at the joined paper, its Materials and Methods (page 11414) describes the method to use to detect by HPLC the fluorescent product obatined with H2O2.The method consists to incubate 5 min at room temperature the reaction assay that may contain H2O2 with homovanillic acid in the presence of horseradish peroxidase. If H2O2 is present, it will react with peroxidase and homovanillic acid to yield quantitatively homovanillic acid dimers that are highly fluorescent (Excitation 315 nm, emmission 425 nm). These dimers can be easily detected on HPLC.
This way you can measure accurately the P450-catalyzed production of H2O2; if it occurs!
Best regards,
Pr Philippe Urban
Dear Bin Guo,
As my colleague Vladimir Mishin recently showed (ToxSci, 07/2014), in the absence of cytochrome P450’s the human recombinant NADPH cytochrome P450 reductase generated low, amounts of H2O2 (~ 0.04 nmole H2O2/min/100 Units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with reductase. CYP1A1 was the most active (6.0 nmoles H2O2/min/nmole P450), followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2 and CYP2C. However, since CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these these enzymes are the major source of H2O2. Directly answering to your question, rough estimations reveal that in each 5 mg of microsomal protein (containing ~ 10 nmol CYP1A1), generation of H2O2 might easily achieve the level above nM.
Best wishes,
Ilya Tsyrlov
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