Fluorescence intensity decays of 1 μM HA-FRET in the absence and presence of HA-ase. The HA-FRET sample (0.25ml) was incubated with 100 U/ml HA-ase for 1 hour. Left: observation at the donor emission. In the presence of HA-ase, the lifetime of the donor increases and the intensity decay becomes less heterogeneous, indicating a release of FRET.
Right: observation at the acceptor emission. In the presence of the HA-ase, the lifetime becomes shorter. In the absence of the HA-ase, the decay is longer and an approximation with a multi-exponential model results in a one weak negative exponent which is characteristic for an excited state reaction.