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

A general method is presented here for the determination of the K-m, k(cat) , and k(cat)/K-m of fluorescence resonance energy transfer (FRET) substrate s using a fluorescence plate reader. A simple empirical method for correcti ng for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters, Inner filter effec t corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measur ement of a gm value of 4.4 +/- 0.3 mu M and k(cat)/K-m value of 96,500 +/- 5800 M-1 s(-1). These values are very close to the HPLC-determined K-m valu e of 4.6 +/- 0.7 mu M and k(cat)/K-m value of 92,600 +/- 14,000 M-1 s(-1). We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of K-i and K-i' values and kine tic inhibition mechanisms for HCV NS3 protease inhibitors. (C) 1999 Academi c Press.