Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes

Abstract

The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.

Figure 1. Fluorescence analysis of H₂O₂ standards using the AR/HRP assay

Panel A. Reactions were initiated by the addition of AR/HRP (arrow). Fluorescence of resorufin was recorded ≈45 sec later. In this calibration experiment, each point represents the mean ± SD (n = 3). The lowest curve represents background and temporal fluorescence changes of blank samples (in the absence of H₂O₂). In most cases, the error bars were less than 5% and fell within the symbols. Panel B. Comparison of the AR/HRP assay and ferrous-thiocyanate assay for analyzing H₂O₂ standards. Upper panels. Analysis of H₂O₂ using the AR/HRP assay. The curve represents the best fit of the average of relative fluorescence units (RFU) for each H₂O₂ standard obtained from three replicates assayed on 6 different days. Each point and error bar on the curve represent the mean ± SD (n = 18). The inset shows the calibration curve at low H₂O₂ concentrations (from 0.0 to 100 pmoles/well). The dashed line on the Y-axis indicates the mean +10 SD’s of blank samples and the dashed line on the x-axis indicates the LLQ value which is 12 pmoles of H₂O₂/well. Lower panels. Analysis of H₂O₂ in the ferrous thiocyanate assay. Each point on the curve represents the mean ± SD of H₂O₂ standards obtained with three replicates assayed on 4 days. OD values were adjusted automatically to 1 cm on the microplate reader using the path check option. The inset shows the calibration curve at low H₂O₂ concentrations (from 0.0 to 1500 pmoles/well). The dashed line on the Y-axis indicates the mean +10 SD’s of blank samples and the dashed line on the x-axis indicates the LLQ value which is 280 pmoles of H₂O₂/well.

Figure 2. Kinetics of H₂O₂ generation by native microsomes

Panels A and C. Continuous recordings of H₂O₂ generation by rat and human liver microsomes. Each well on a 96 well plate was loaded with 90 μl of incubation media containing: potassium phosphate buffer, 50 mM, pH 7.7, 1 mM DETAPAC, 1 mM azide, 500 Units/ml SOD, 0.1 mM NADPH, and an NADPH regenerating system, (10.0 mM glucose-6-phosphate and 0.5 Units/ml glucose-6- phosphate dehydrogenase). The plate was preincubated at 37°C for 5 min in the plate reader chamber. The wells were then loaded with 50 μl of AR/HRP. The reactions were started by the addition of 10 μl microsomal preparations using a multichannel pipette (as indicated by arrow). The lines represent time-dependent changes of fluorescence generated with different amounts of microsomal protein (as indicated). The data for rats are the mean ± SD of two experiments and humans, are the mean ± SD of three experiments. Each microsomal protein concentration was done in triplicate. Panel B. H₂O₂ generation by rat liver microsomes assayed at fixed time points. Mixes were prepared as indicated above; reactions were initiated by the addition of 10 μg of microsomal protein. In this mode of assay, the 50 μl of AR/HRP mixture were added to the wells at the indicated time points. The bars reflect the fluorescence intensity recorded at those time points. The dashed line connecting the top of the bars was generated by GraphPad Prism and used for calculating the absolute rates of H₂O₂ production.

Figure 3. Kinetics of H₂O₂ generation by recombinant human microsomal enzymes

Assays mixes were prepared as described in the legend to Figure 2. Experiments were performed with a mixture of human drug metabolizing cytochrome P450’s coexpressed with human NADPH-cytochrome P450 reductase. Panel A. Continuous recording of H₂O₂ generation by human microsomal enzymes. Each well contained 10 μg/well (12 Units of NADPH-cytochrome P450 reductase, 1.5 pmol cytochrome P450) of protein. The data are the mean ± SD of three separate experiments in duplicate. Panel B. H₂O₂ generation by recombinant human microsomal enzymes at fixed time points. The data are the mean ± SD of two experiments with each microsomal protein concentration analyzed in triplicate. The dashed lines connecting the tops of the bars were generated by GraphPad Prism and were used for calculation of absolute rates of H₂O₂ production.

Figure 4. Kinetics of H₂O₂ generation by recombinant recombinant human NADPH-cytochrome P450 reductase

Assay mixes were prepared as described in the legend to Figure 3. Panel A. Continuous recording of H₂O₂ generation. The amounts of microsomal protein are indicated as Units of NADPH-cytochrome P450 reductase activity in cytochrome c reduction. The data are the mean ± SD of four experiments with each microsomal protein concentration analyzed in duplicate. Panel B. H₂O₂ generation by human NADPH-cytochrome P450 reductase assayed at fixed time points. The amounts of microsomal protein added were: open bars - 6 Units of NADPH-cytochrome P450 reductase/well; dotted bars - 12 Units of NADPH-cytochrome P450 reductase/well. The data are the mean ± SD of four separate experiments performed in duplicate. The dashed lines connected tops of the bars were generated by GraphPad Prism and used for calculation of absolute rates of H₂O₂ production. Panel C. Assay of H₂O₂ generation in the presence of 50 μM paraquat in continuous (circles) and fixed time points mode (black bars) assays. The amounts of microsomal protein added to the wells were 0.2 Units of NADPH-cytochrome P450 reductase/well. Bottom line (triangle) indicates the level of background fluorescence in wells with no proteins added. The data are the mean ± SD of two separate experiments performed in triplicate.