A new protease activity assay using fluorescence polarization

Abstract

Fluorescence polarization (FP) technology was used to develop an assay for protease activity that is more sensitive than other nonradioactive protease assays and requires no separations, precipitations or transfers of the reaction mixture. FP measures changes in the molecular volume of fluorescently labeled molecules. In the assay described in the present studies, changes in molecular volume due to the cleavage of intact fluorescein thiocarbamoyl (FTC)-casein molecules to smaller FTC peptides were measured. The sensitivity of the FP protease assay was compared with a non-FP fluorescent assay. The FP-based assay was twofold to twentyfold more sensitive than the non-FP assay, depending on the protease tested. Because measurements were taken in real time, the progress of the reaction was followed both kinetically and at a single time point. This assay provided a sensitive measure of activity for the three common protease classes: serine proteases, sulfhydryl proteases and acid proteases.