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

This paper presents the application of fluorescence polarization to the det ermination of dissociation constants for competitive inhibitors that bind t o enzymes. This steady-state enzyme kinetic study measures the inhibition o f the conversion of a fluorescently tagged substrate to a lower molecular w eight fluorescent product by calpain II. It relies on the measurement of a parameter proportional to velocity, which is sufficient for this type of an alysis. The strengths and limitations of the method are discussed. Inhibiti on constants for filamin and spectrin determined by this method are 125 nM and 13 nM respectively. (C) 1999 Federation of European Biochemical Societi es.