Fluorescence-binding studies of full-length Xenopus IRBP to all- trans retinol. The concentration of Xenopus IRBP was 0.65 μM in each panel. A) Titrations of IRBP with all-trans retinol as followed by monitoring the increase in retinol fluorescence (excitation, 330 nm; emission, 480 nm). Retinol fluorescence in the presence of IRBP (○,◊, Δ) is compared with that in the presence of an fluorescence matched solution of N-acetyl-L-tryptophanamide (□). The difference between these two curves, the fluorescence enhancement (-●-), represents all-trans retinol bound to the protein. The curve is a nonlinear least squares fit of Equation 1 to the binding data. Error bars are too small to be visualized. The number of binding sites per molecule of protein (N) was 3.19 ± 0.10 with K
all-trans= 0.30 ± 0.05 μM (standard error of the mean). B). Emission spectra of apo- and holo-IRBP (curves a and b, respectively) were obtained upon excitation at 280 nm in the presence of a 10 fold excess of all-trans retinol. The drop in emission at 340 nm represents quenching of the protein's intrinsic fluorescence. The emission at 480 (arrow) represents energy transfer to the bound retinol. C) Titration monitoring quenching of intrinsic protein fluorescence by bound retinol. Excitation and emission wavelengths were 280 and 340 nm, respectively. The inner filter effect has been accounted for graphically as previously described (50) (-○-, actual meausurements; -●-, after correction). Calculated binding parameters: N = 1.93 ± 0.41; K
all-trans= 0.66 ± 0.14 μM. D) Titration monitoring energy transfer (increase in fluorescence at 480 (arrow). Calculated binding parameters: N = 3.72 ± 0.20; K
all-trans= 0.29 ± 0.12.