Figure 4.

P1 is fluorogenic upon binding and reacting with RA to form a covalent conjugate. P1 (5 μM) was incubated with RA (5 μM) or the K210A RA mutant (5 μM) for 24 h in buffer at 25 °C resulting in complete covalent modification of RA, but not the K210A mutant which forms a non-covalent K210A RA·P1 complex. P1 (5 μM) is dark in buffer and is only very weakly fluorescent when bound to the K210A RA mutant. In contrast, P1 is strongly fluorescent upon forming a covalent conjugate with RA. Excitation and emission spectra were recorded using an Aviv fluorescence spectrometer. Samples in the inset were photographed under illumination with a hand-held UV lamp.