Application of fluorescence polarization to the steady-state enzyme kinetic analysis of calpain II.

This paper presents the application of fluorescence polarization to the determination of dissociation constants for competitive inhibitors that bind to enzymes. This steady-state enzyme kinetic study measures the inhibition of the conversion of a fluorescently tagged substrate to a lower molecular weight fluorescent product by calpain II. It relies on the measurement of a parameter proportional to velocity, which is sufficient for this type of analysis. The strengths and limitations of the method are discussed. Inhibition constants for filamin and spectrin determined by this method are 125 nM and 13 nM respectively.