Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes.
Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes.
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DOI：
10.1016/j.freeradbiomed.2010.02.030
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发表时间：
2010-06-01
影响因子：
7.4
通讯作者：
Laskin, Jeffrey D.
中科院分区：
文献类型：
--
作者：
Mishin, Vladimir;Gray, Joshua P.;Heck, Diane E.;Laskin, Debra L.;Laskin, Jeffrey D.
通过细胞色素P450单加氧酶系统形成活性氧被认为是由于NADPH-细胞色素P450还原酶的自氧化和氧结合的细胞色素P450中间体的非生产性衰变。为了表征重组微粒体酶中的这一过程，我们使用了基于Amplex-Red氧化的高灵敏度过氧化氢测定法。该检测试剂盒的灵敏度（LLD = 5.0 pmole/检测试剂盒，LLQ = 30 pmole/检测试剂盒）比标准硫氰酸亚铁检测试剂盒的灵敏度高20倍。我们发现重组人NADPH-细胞色素P450还原酶复合物（0.034 nmol过氧化氢/min/100单位NADPH-细胞色素P450还原酶）自发产生的过氧化氢较低，但可检测到。当重组细胞色素P450酶与NADPH-细胞色素P450还原酶共表达时，观察到显著更高的过氧化氢产生速率（0.31纳摩尔过氧化氢/分钟/100单位的NADPH-细胞色素P450还原酶）。这是独立的添加任何外源性细胞色素P450底物。这些数据表明，细胞色素P450是重组细胞色素P450单加氧酶系统中过氧化氢的主要来源。此外，细胞色素P450产生活性氧物质不需要底物结合。
The formation of reactive oxygen species by the cytochrome P450 monoxygenase system is thought to be due to autooxidation of NADPH-cytochrome P450 reductase and the non-productive 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 = 5.0 pmoles/assay, and LLQ = 30 pmoles/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 (0.034 nmoles hydrogen peroxide/min/100 Units 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 (0.31 nmoles of hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450’s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450’s to generate reactive oxygen species.
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影响因子：
4.1
作者：
BALVERS, WG;BOERSMA, MG;RIETJENS, IMCM
通讯作者：
RIETJENS, IMCM
影响因子：
3.9
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HILDEBRANDT, AG;ROOTS, I
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ROOTS, I
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通讯作者：
LYAKHOVICH, VV