Abstract

A sensitive fluorescence assay based on Amplex Red (AR) oxidation by horseradish peroxidase (AR/HRP) is described which continuously monitor rates of H₂O₂ production by microsomal enzymes in the presence of relatively high concentrations of NADPH. NADPH and NADH are known to interact with HRP and generate significant quantities of superoxide anion, a radical that spontaneously dismutates to form H₂O₂ which interferes with the AR/HRP assay. Microsomal enzymes generate H₂O₂ as a consequence of electron transfer from NADPH to cytochrome P450 hemoproteins with subsequent oxygen activation. We found that superoxide anion formation via the interaction of NADPH with HRP was inhibited by superoxide dismutase (SOD) without affecting H₂O₂ generation by microsomal enzymes. Using SOD in enzyme assays, we consistently detected rates of H₂O₂ production using microgram quantities of microsomal proteins (2.62 ± 0.20 picomol/min/µg protein for liver microsomes from naïve female rats, 12.27 ± 1.29 for liver microsomes from dexamethasone induced male rats, and 2.17 ± 0.25 picomol/min/µg protein for human liver microsomes). This method can also be applied to quantify rates of H₂O₂ production by oxidases where superoxide anion generation by NADH or NADPH and HRP can interfere with enzyme assays.

摘要

描述了一种基于辣根过氧化物酶 (AR/HRP) 对 Amplex Red (AR) 氧化的灵敏荧光测定，该测定在存在相对高浓度的 NADPH 的情况下连续监测微粒体酶产生 H 2 _O 2_的速率。已知 NADPH 和 NADH 与 HRP 相互作用并产生大量超氧阴离子，超氧阴离子是一种自发歧化形成 H ₂ O ₂的自由基，会干扰 AR/HRP 测定。由于电子从 NADPH 转移到细胞色素 P450 血红素蛋白以及随后的氧活化，微粒体酶产生 H ₂ O _{2 。}我们发现超氧化物歧化酶(SOD) 抑制NADPH 与HRP 相互作用形成的超氧阴离子，但不影响微粒体酶产生H ₂ O _{2 。}在酶测定中使用 SOD，我们使用微克量的微粒体蛋白一致地检测了 H 2 O 2 的产生率（对于幼稚雌性大鼠的肝微粒体为 2.62 ± 0.20 皮摩尔/分钟/μg 蛋白，对于来自地塞米松诱导的雄性大鼠的肝微粒体为_12.27 ± _1.29大鼠，以及人肝微粒体 2.17 ± 0.25 皮摩尔/分钟/μg 蛋白质）。该方法还可用于量化氧化酶产生的 H ₂ O ₂速率，其中 NADH 或 NADPH 和 HRP 产生的超氧阴离子会干扰酶测定。