Kinetics of amyloid β-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays

Proteases that degrade the amyloid β-protein (Aβ) are important regulators of brain Aβ levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Aβ degradation assays based on the novel substrate, fluorescein-Aβ-(1-40)-Lys-biotin (FAβB). Liquid chromatograpby/mass spectrometric analysis shows that FAβB is hydrolyzed at closely similar sites as wild-type Aβ by neprilysin and insulin-degrading enzyme, the two most widely studied Aβ-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAβB degradation by several Aβ-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Aβ-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.