Fluorescence Polarization Assays for the Detection of Proteases and Their Inhibitors

Abstract

Fluorescence polarization (FP) is a powerful technology for the determination of the sizes of molecules in solution and has been extensively used in studies of the binding of small ligands to their receptors. Alternatively, it can be used to monitor the conversion of large molecules to small ones, for example, in studies of hydrolytic enzymes such as proteases, DNases, RNases, and carbohydratases. Proteases are becoming increasingly important targets of drug discovery in the fields of infectious diseases, inflammatory disorders, and apoptosis, among others. FP has been shown to be an extremely useful assay technology for the nonspecific and specific detection and measurement of proteases and their inhibitors. Although fluorescein is the fluorophore most often used for FP, it suffers from the disadvantages of being essentially nonfluorescent below pH 6, where many proteases of interest operate, and derivatives are not available in the red portion of the spectrum, where very few interferences are encountered. The development of highly sensitive, pH-independent assays using BODIPY(r)*-labeled a-caseins at fluorescein and Texas Red wavelengths are described. These assays can detect less than 1 ng/ml of pepsin, at pH 2.0, and less than 10 ng/ml trypsin, at pH 7.4, with a 5-min incubation time. Using 1 ttg/ml trypsin and BODIPY(r) TR-X-labeled cv-casein, soybean trypsin inhibitor (STI) can readily be detected at less than 50 ng/ml with a 5-min incubation time. Since these assays require a single, stable reagent in a homogeneous format, they readily lend themselves to the high throughput screening of compound libraries for protease inhibitors.