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Application of fluorescence polarization to the steady‐state enzyme kinetic analysis of calpain II
Author(s) -
Sem Daniel S.,
McNeeley Patricia A.
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01655-x
Subject(s) - spectrin , calpain , fluorescence , chemistry , fluorescence anisotropy , enzyme , dissociation constant , kinetic energy , steady state (chemistry) , biophysics , analytical chemistry (journal) , kinetics , chromatography , biochemistry , biology , physics , membrane , optics , cytoskeleton , receptor , quantum mechanics , cell
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.