Analysis of Peroxidases Using Liquid Chromatography, Post-Column Substrate Conversion and Fluorescence Detection

Rob Haselberg, C.M. Hempen, S.M. van Leeuwen, M. Vogel, U. Karst

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    A liquid chromatographic method with on-line activity determination for microperoxidases has been developed. After enzymatic digestion of a cytochrome, possibly under formation of microperoxidases, the product mixture is separated by reversed-phase liquid chromatography. The products first pass a diode-array detector, and are then subjected to a reaction with 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) and hydrogen peroxide. In a reaction coil, microperoxidases catalyze the reaction under formation of the fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA). Quantification of the microperoxidases is performed using a fluorescence detector at an excitation wavelength of 470 nm and an emission wavelength of 545 nm, respectively. For this LC-based detection system, limits of detection are 3 × 10−8 mol/L, limits of quantification are 9 × 10−8 mol/L, and a linear range from 9 × 10−8 mol/L to 3 × 10−6 mol/L is obtained for the microperoxidases MP-9 and MP-11. A highly active microperoxidase MP-6 was found in the reaction of cytochrome c from bovine heart with protease from streptomyces griseus.
    Original languageUndefined
    Pages (from-to)47-53
    Number of pages7
    JournalJournal of chromatography. B: Analytical technologies in the biomedical and life sciences
    Issue number1
    Publication statusPublished - 2006


    • MS detection
    • METIS-231338
    • Post-column derivatization
    • Microperoxidases
    • Digestion
    • HPLC
    • Fluorescence detection
    • IR-59868

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