Figure 8.

11-cis retinaldehyde binding to full-length Xenopus IRBP. Since retinaldehydes are nonfluorescent compounds their binding cannot be followed by ligand fluorescence enhancement or energy transfer. Here, the binding of 11-cis retinaldehyde is followed by monitoring quenching of endogenous protein fluorescence, and indirectly by competition with the efficient fluorophore all-trans retinol. A) Representative titrations monitoring quenching of intrinsic protein fluorescence by bound 11-cis retinaldehyde. Excitation and emission wavelengths were 280 and 340 nm, respectively. The inner filter effect was accounted for by graphical correction as previously described [56] (-●-, before correction for inner filter effect; -●-, after correction). The binding parameters were calculated to be: N = 1.81 ± 0.15; K_d^11-cis = 0.28 ± 0.05 μM. B. Representative competition titration. From each titration the emission of an 0.D.₂₈₀ matched solution of N-acetyl-L-tryptophanamide was subtracted. Binding parameters: N = 3.53 ± 0.19 with K_d^all-trans = 0.22 ± 0.13 μM; K_d^11-cis = 0.21 ± 0.10 μM.