A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases. • SYPRO Orange is employed to monitor degradation of native proteins in real time. • A real-time PCR instrument is used for continuous fluorescence measurements. • The assay is suitable for a broad range of temperatures. • The assay is compatible with a low-volume, high-throughput format.