The Scopoletin-HRP Fluorimetric Determination of H2O2 in Seawaters—A Plea for the Two-Stage Protocol
| Autor: | Yao-Chu Wu, Man Wu, George T.F. Wong |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
010504 meteorology & atmospheric sciences
QH301-705.5 hydrogen peroxide 010502 geochemistry & geophysics 01 natural sciences Biochemistry Genetics and Molecular Biology (miscellaneous) Horseradish peroxidase Article Reaction rate chemistry.chemical_compound Structural Biology Scopoletin Fluorometer fluorimetric determination scopoletin horseradish peroxidase Biology (General) Hydrogen peroxide 0105 earth and related environmental sciences Chromatography biology Chemistry Fluorescence Photobleaching biology.protein Stoichiometry Biotechnology |
| Zdroj: | Methods and Protocols Methods and Protocols, Vol 1, Iss 1, p 4 (2017) Methods and Protocols; Volume 1; Issue 1; Pages: 4 |
| ISSN: | 2409-9279 |
| Popis: | A single solution protocol has been widely used for the fluorimetric determination of H2O2 in natural waters by its bleaching of the fluorescing scopoletin in the presence of the enzyme horseradish peroxidase (HRP). In this protocol, the reaction between scopoletin and H2O2 in the sample and the subsequent internal additions, and the measurements of the fluorescence are all carried out at a single pH in a fluorometer cell. It is found that this protocol is prone to four sources of possible error. The variability in the reaction stoichiometry between scopoletin and H2O2 in the presence of varying amounts of excess scopoletin, the effect of pH on the rate of reaction between scopoletin and H2O2, the photobleaching of scopoletin, and the de-activation of HRP. These possible sources of error can be circumvented in a two-stage protocol in which the reaction between H2O2 and scopoletin is carried out immediately upon sampling at a pH of 7, and the measurement of the fluorescence is carried out later on at a pH of 9. It should be the protocol of choice. Furthermore, in the two-stage protocol, after the initial reaction between H2O2 and scopoletin, the sample may be stored at room temperature for six days and at 4 °C for at least a month before its fluorescence is measured. This option can significantly reduce the logistics in the field. |
| Databáze: | OpenAIRE |
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