Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection

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Haselberg, Rob and Hempen, Christel and Leeuwen, Suze M. van and Vogel, Martin and Karst, Uwe (2007) Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection. Journal of Chromatography B, 830 (1). pp. 2629-2637. ISSN 1570-0232

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Abstract: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.
Item Type:Article
Copyright:© 2005 Elsevier
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Link to this item:http://purl.utwente.nl/publications/59868
Official URL:http://dx.doi.org/10.1016/j.jchromb.2005.10.013
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