Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10536007 | Analytical Biochemistry | 2007 | 9 Pages |
Abstract
Fluorescence change is convenient for monitoring enzyme kinetics. Unfortunately, it loses linearity as the absorbance of the fluorescent substrate increases with concentration. When the sum of absorbance at excitation and emission wavelengths exceeds 0.08, this inner filtering effect (IFE) alters apparent initial velocities, Km, and kcat. The IFE distortion of apparent initial velocities can be corrected without doing fluorophore dilution assays. Using the substrate's extinction coefficients at excitation and emission wavelengths, the inner filter effect can be modeled during curve fitting for more accurate Michaelis-Menten parameters. A faster and simpler approach is to derive kcat and Km from progress curves. Strategies to obtain reliable and reproducible estimates of kcat and Km from only two or three progress curves are illustrated using matrix metalloproteinase 12 and alkaline phosphatase. Accurate estimates of concentration of enzyme-active sites and specificity constant kcat/Km (from one progress curve with [S] âªÂ Km) confer accuracy, freedom of choices of [S], and robustness to kcat and Km globally fitted to a few progress curves. The economies of the progress curve approach make accurate kcat and Km more accessible from fluorescence measurements.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Mark O. Palmier, Steven R. Van Doren,