1. Dissolve 20 mg TOOS with 10 ml PBS to prepare 6.6 mM TOOS solution.
2. Dissolve 14 mg 4-aminoantipyrin (AA) with 10 ml PBS to prepare 6.6 mM 4-AA solution.
3. Prepare 2 U/ml horseradish peroxidase solution with PBS.
4. Mix the same volume of each solution together to prepare assay solution. Store the assay solution at 4°C with protection from light.
1. Prepare sample solutions for the enzymatic oxidation reaction. The pH range of the buffer solution should be from 5.5-9.5.
2. Prepare standard solutions containing known amounts of substrate using the same buffer.
3. Add the appropriate units of oxidase to the sample solution, followed by an addition of the same volume of the assay solution.
4. Incubate the mixture at room temperature or at 37oC for 30 min to 1 hour.
5. Measure the O.D. at 555 nm.
6. Prepare a standard curve, and determine the substrate concentration in the sample solution.
1. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Photometric Determination of Hydrogen Peroxide. II. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline derivatives. Chem Pharm Bull. 1982;30:2492-2497.
2. K. Tamaoku, et al., New water-soluble Hydrogen Donors for the Enzymatic Spectrophotometric Determination of Hydrogen Peroxide. Anal Chim Acta. 1982;136:121-127.
3. B. C. Madsen, et al., Flow Injection and Photometric Determination of Hydrogen Peroxide in Rainwater with N-Ethyl-N-(sulfopropyl)aniline sodium salt. Anal Chem. 1984;56:2849-2850.