Determination of CoA-HNE binding and off-rate. A. Quenching of tryptophan fluorescence as a function of the binding of inhibitor CoA-HNE by recombinant GST-bfAANATα. Binding was not observed in the presence of 4 M guanidine, which denatures the protein. B. Off-rate of CoA-HNE from the active site of the enzyme was measured by displacement with another inhibitor. Fluorescence anisotropy is shown as a function of time. Samples of proteins (2 μM) were 90% saturated with CoA-HNE. After 5 min the fluorescence anisotropy was measured. At t = 200 s, CoA-T was added to a final concentration of 170 μM and fluorescence anisotropy measurement was resumed. oAANAT data from a previous study are included for comparison . Data are presented as the mean ± S.E.M (N = 3). For further details see Methods.