Difference between revisions of "Towne 2004 Anal Biochem"
| Line 9: | Line 9: | ||
}} | }} | ||
{{Labeling | {{Labeling | ||
|instruments= | |instruments=Spectrofluorometry | ||
|injuries=RONS; Oxidative Stress | |injuries=RONS; Oxidative Stress | ||
|preparations=Enzyme | |preparations=Enzyme | ||
}} | }} | ||
Revision as of 13:44, 18 January 2013
| Towne V, Will M, Oswald B, Zhao Q (2004) Complexities in horseradish peroxidase-catalyzed oxidation of dihydroxyphenoxazine derivatives: appropriate ranges for pH values and hydrogen peroxide concentrations in quantitative analysis. Anal Biochem 334, 290-296. |
» http://www.sciencedirect.com/science/article/pii/S0003269704006499
Towne V, Will M, Oswald B, Zhao Q (2004) Anal Biochem
Abstract: 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 • Keywords: Amplex red, Horseradish peroxidase, Hydrogen peroxide
Labels:
Stress:RONS; Oxidative Stress"RONS; Oxidative Stress" is not in the list (Cell death, Cryopreservation, Ischemia-reperfusion, Permeability transition, Oxidative stress;RONS, Temperature, Hypoxia, Mitochondrial disease) of allowed values for the "Stress" property.
Preparation: Enzyme
HRR: Spectrofluorometry"Spectrofluorometry" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
