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

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Microperoxidases are crucial enzymatic components.
• Accurate quantification of microperoxidases is essential for biochemical research.
• Existing methods may lack sensitivity or on-line capabilities.

Purpose of the Study:

• To develop a novel liquid chromatographic method for on-line activity determination of microperoxidases.
• To establish sensitive and quantitative detection limits for microperoxidases.
• To identify microperoxidases formed during enzymatic digestion of cytochrome c.

Main Methods:

• Reversed-phase liquid chromatography (RPLC) for separation of digestion products.
• On-line reaction with 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) and hydrogen peroxide.
• Fluorescence detection at specific excitation (470 nm) and emission (545 nm) wavelengths for MNBDA.

Main Results:

• Established a sensitive LC-based detection system for microperoxidases.
• Achieved limits of detection (3 x 10(-8) mol/L) and quantification (9 x 10(-8) mol/L).
• Demonstrated a linear quantification range from 9 x 10(-8) to 3 x 10(-6) mol/L for MP-9 and MP-11.
• Identified a highly active microperoxidase MP-6 in cytochrome c digestion.

Conclusions:

• The developed LC method provides sensitive and accurate on-line quantification of microperoxidases.
• This method is valuable for studying microperoxidase formation and activity.
• The findings contribute to understanding enzymatic processes involving cytochromes.