Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection
| Autor: | Christel Hempen, Martin Vogel, Rob Haselberg, Suze M. van Leeuwen, Uwe Karst |
|---|---|
| Přispěvatelé: | BioAnalytical Chemistry, AIMMS |
| Rok vydání: | 2005 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Electrospray ionization microperoxidases Clinical Biochemistry post-column derivatization Analytical chemistry digestion Biochemistry High-performance liquid chromatography Post-column derivatization Sensitivity and Specificity Fluorescence spectroscopy Fluorescence detection IR-59868 Analytical Chemistry Column chromatography Animals Detection limit Chromatography Microperoxidases Chemistry Myocardium Substrate (chemistry) Cytochromes c MS detection Cell Biology General Medicine Hydrogen-Ion Concentration METIS-231338 Fluorescence Spectrometry Fluorescence Linear range Peroxidases fluorescence detection Digestion Cattle HPLC Chromatography Liquid |
| Zdroj: | Journal of chromatography. B: Analytical technologies in the biomedical and life sciences, 830(1), 47-53. Elsevier Journal of Chromatography B, 830(1), 47-53. Elsevier Haselberg, R, Hempen, C M, van Leeuwen, SM, Vogel, M & Karst, U 2006, ' Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection ', Journal of Chromatography B, vol. 830, no. 1, pp. 47-53 . https://doi.org/10.1016/j.jchromb.2005.10.013 |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2005.10.013 |
| Popis: | 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 x 10(-8) mol/L, limits of quantification are 9 x 10(-8) mol/L, and a linear range from 9 x 10(-8) mol/L to 3 x 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect