Evaluation of the horseradish peroxidase-scopoletin method for the measurement of hydrogen peroxide formation in biological systems

✍ Scribed by A. Boveris; E. Martino; A.O.M. Stoppani

• Publisher: Elsevier Science
• Year: 1977
• Tongue: English
• Weight: 747 KB
• Volume: 80
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(77)90634-0

✦ Synopsis

Using the glucose oxidase system as the source of H,O,, the accuracy and reliability of the horseradish peroxidase (HRP)-scopoletin method for measuring HZ02 were critically evaluated. The rates of scopoletin oxidation and of formation of the HRP-HZ02 Compound II intermediate were measured and compared with those obtained by the cytochrome c peroxidase (CCP) method or with those calculated from the oxygen uptake corresponding to the H,O, produced by the glucose oxidase system. Determinations were made of both the optimal concentrations of HRP and scopoletin necessary to adequately couple the production and utilization of Hz02 and of the kinetic parameters of the overall reaction. In the absence of external hydrogen donors for the HRP reaction, scopoletin oxidation and formation of the intermediate HRP-H,O1 Compound II yielded 70 and 80%, respectively, of the H,O, generated. The interference of catalase in the HRP-scopoletin assay was evaluated in terms of the catalase/ HRP heme ratio. A simple procedure is given to correct the rates of HZO1 measured by the HRP-scopoletin assay for catalase interference, according to the heme ratio. The HRP-scopoletin assay was applied to a series of mitochondrial preparations including rat heart, pigeon heart, rat liver, mung bean, and yeast mitochondria, and the results are compared to those obtained by the CCP method. The effectiveness of the HRP-scopoletin assay ranged between 2 and 60% of that of the CCP assay. The difference is, in all likelihood, due to the broad specificity of the HRP intermediate which accepts electrons from donors present in biological preparations.