Studies on the Fluorescence of the Human Vitamin A-transporting Plasma Protein Complex and Its Individual Components

The fluorescence properties of the human prealbumin-retinol-binding protein (RBP) complex and of its individual components are described. At neutral pH, RBP and the protein complex have two fluorescence bands: one at 335 nm and the other, associated with the retinol, at 470 nm. Prealbumin has only a fluorescence at 335 nm (quantum yield 0.11). The quantum yield of retinol increased by 50% and the emission spectrum was blue shifted on RBP forming a complex with prealbumin. RBP, at low ionic strength where the binding of RBP and prealbumin is abolished, exhibited a decrease in the quantum yield of retinol. With the protein fluorescence of RBP and the fluorescence of its cofactor, it was also possible to show the occurrence of transfer of excitation energy from the protein moiety to the retinol (efficiency of transfer: 60%). Studies on the accessibility of the aqueous medium (or protons) to the retinol site revealed that on complex formation between RBP and prealbumin most of the retinol-solvent interactions are abolished, suggesting that one of the functions of prealbumin is to stabilize the retinol-binding site of RBP. The estimated apparent association constant is 2 x 107 m-1 for the interaction of retinol-containing RBP and prealbumin. The binding constant of prealbumin and vitamin A-free RBP was also determined. A similar value was found and it is thus concluded that retinol has no major influence on the tertiary structure of RBP.