Rapid screening of proteolytic enzymes and their inhibitors using fluorescence methods

New fluorescence methods for the determination of proteolytic enzymes and their inhibitors have been investigated. Fluorescence polarisation and energy transfer methods have both been examined and of these two approaches energy transfer methods have proven superior. Out of the energy transfer methods, the use of an over-labelled substrate, which is virtually non-fluorescent but becomes highly fluorescent upon proteolytic liberation of dye-labelled peptides, has been demonstrated to be most successful. A substrate utilising α-casein labelled with a long-wavelength BODIPY® dye has been synthesised and shown to be a suitable intramolecularly quenched conjugate for the determination of proteolytic enzymes and their inhibitors in flow-based screening programmes. [Continues.]