Figure 3.

Ligand binding studies with the purified lipocalin variant FluA. (A) Fluorescence titration (λ_Ex = 280 nm; λ_Em = 340 nm) of a 1 μM protein solution with fluorescein (■), 4-aminofluorescein (●), 4-glutarylamidofluorescein (▴), pyrogallol red (□), phenolphthalein (○), and rhodamine B (▵). The data were corrected and fitted as described in Materials and Methods. The resulting K_d values are given in Table 2. In the case of fluorescein the curve (dotted line) could not be fitted as perfectly as with the other ligands. (B) Fluorescence titration (λ_Ex = 490 nm; λ_Em = 512 nm) of a 1 μM fluorescein solution with FluA (■) or with BBP (×). The data for FluA were fitted without correction, yielding K_d = 35.2 ± 3.2 nM.