Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase.

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.