Towne Victoria, Will Mark, Oswald Brent, Zhao Qinjian
Department of Bioprocess and Bioanalytical Research, Merck Research Laboratories, West Point, PA 19486, USA.
Anal Biochem. 2004 Nov 15;334(2):290-6. doi: 10.1016/j.ab.2004.07.037.
The highly sensitive, convenient fluorescence assay, based on the oxidation of nonfluorescent 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to highly fluorescent resorufin, is becoming increasingly popular for hydrogen peroxide quantitation. Yet, the intricacies of the horseradish peroxidase-catalyzed oxidation of the reductant substrate Amplex Red by hydrogen peroxide and the resulting resorufin could complicate the assay design and data interpretation. In particular, substrate inhibition and enzyme inactivation at higher hydrogen peroxide concentrations were known to affect the enzyme kinetics and end-point fluorescence. In addition, here we report the spontaneous transformation of resorufin to less or nonfluorescent product(s) in the absence of hydrogen peroxide and horseradish peroxidase. This spontaneous decay of resorufin fluorescence is most prominent in the pH range 6.2-7.7, likely due to general base-catalyzed de-N-acetylation and polymerization of resorufin. From a practical point of view, precautions for properly designing assays for hydrogen peroxide or characterizing hydrogen peroxide-generating systems are discussed based on the spontaneous transformation of resorufin to less fluorescent compound(s), substrate inhibition and enzyme inactivation at higher (>100 microM) hydrogen peroxide concentrations, and enzymatic oxidation of resorufin to nonfluorescent resazurin.
基于将无荧光的10-乙酰基-3,7-二羟基吩恶嗪（Amplex Red）氧化为高荧光的试卤灵的高灵敏度、便捷的荧光测定法，在过氧化氢定量分析中越来越受欢迎。然而，辣根过氧化物酶催化过氧化氢氧化还原底物Amplex Red并生成试卤灵的复杂过程，可能会使测定设计和数据解读变得复杂。特别是，已知在较高过氧化氢浓度下的底物抑制和酶失活会影响酶动力学和终点荧光。此外，我们在此报告，在没有过氧化氢和辣根过氧化物酶的情况下，试卤灵会自发转化为荧光较弱或无荧光的产物。试卤灵荧光的这种自发衰减在pH值6.2 - 7.7范围内最为明显，这可能是由于一般碱催化的试卤灵脱N-乙酰化和聚合反应。从实际角度出发，基于试卤灵自发转化为荧光较弱的化合物、在较高（>100 microM）过氧化氢浓度下的底物抑制和酶失活，以及试卤灵酶促氧化为无荧光的刃天青，讨论了正确设计过氧化氢测定法或表征过氧化氢生成系统的注意事项。
