Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection.
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.
Haselberg
Rob
R
University of Twente, Chemical Analysis Group and MESA+ Institute for Nanotechnology, Enschede, The Netherlands.
Hempen
Christel
C
van Leeuwen
Suze M
SM
Vogel
Martin
M
Karst
Uwe
U
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D045304:Cytochromes c / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000201:enzymology; D010544:Peroxidases / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D045304:Cytochromes c; D010544:Peroxidases; C021799:microperoxidase