Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction

Area of Science:

• Biochemistry
• Enzymology
• Pharmacology

Background:

• Enzyme kinetics are crucial for understanding enzyme function and drug development.
• Accurate measurement of kinetic parameters like Km, kcat, and kcat/Km is essential.
• Fluorescence resonance energy transfer (FRET) substrates are widely used but can be affected by the inner filter effect.

Purpose of the Study:

• To present a general method for determining kinetic parameters (Km, kcat, kcat/Km) of FRET substrates using a fluorescence plate reader.
• To introduce a simple empirical method for correcting the inner filter effect.
• To enable accurate and undistorted kinetic measurements for enzyme inhibitors.

Main Methods:

• Utilized a fluorescence plate reader for FRET substrate analysis.
• Applied a novel empirical method to correct for the inner filter effect.
• Measured hydrolysis rates of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at varying concentrations.

Main Results:

• Achieved accurate determination of Km and kcat/Km values for HCV NS3 protease.
• Obtained Km = 4.4 ± 0.3 μM and kcat/Km = 96,500 ± 5800 M⁻¹s⁻¹, closely matching HPLC-determined values.
• Demonstrated the utility of inner filter effect correction for rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms.

Conclusions:

• The developed method provides accurate and reliable kinetic parameter determination for FRET substrates.
• Inner filter effect correction is critical for undistorted measurements in fluorescence-based assays.
• This method facilitates rapid assessment of enzyme inhibitors, including those targeting HCV NS3 protease.