Interactions of All-trans-, 9-, 11-, and 13-cis-retinal, All-trans-retinyl Acetate, and Retinoic Acid with Human Retinol-binding Protein and Prealbumin

Abstract

The reconstitution of analogs of the native chromophore retinol (vitamin A) with apo-retinol-binding protein (apo-RBP) from human plasma was studied by chromatographic and spectroscopic techniques. All-trans- and 9-, 11-, and 13-cis-retinal combined with apo-RBP in a 1:1 molar ratio while only about 0.85 mole of all-trans-retinoic acid and 0.75 mole of retinyl acetate combined with 1.0 mole of apo-RBP. The various chromophores all bound to the same site on retinol-binding protein and were competitive inhibitors of each other's binding. Upon reconstitution the absorption peak of the chromophore shifted some 9 to 12 nm to the red with the retinal chromophores, 5 nm to the red with retinyl acetate, and 15 nm to the blue with retinoic acid (at pH 9). All the chromophore-RBP complexes showed an induced (extrinsic) Cotton effect of the chromophore absorption band with a rotatory strength of the same order of magnitude as that of the retinol isomers-RBP. Similar to the behavior of the free chromophore in aqueous detergent solutions, the absorption and CD spectra of retinoic acid-RBP were pH dependent, with both the absorption and CD chromophore peak decreasing in magnitude and shifting to the red upon a decrease in pH. The reconstitution of apo-RBP with the various chromophores led to a marked (more than 90%) quenching of the fluorescence of the inherent protein chromophores. Increasing the ionic strength of a retinal-RBP complex led to a shift of the absorption to the blue (7 nm with the 9-cis-retinal), a small decrease in the size of the absorption band, and marked decrease (up to 39% with 11-cis-retinal) of the rotatory strength. Increasing the ionic strength of retinoic acid-RBP solution led to a shift of the absorption peak to the red of about 1 nm, a 2.4% increase in the absorption band, and an increase of about 1% in the rotatory strength. The same change in ionic strength had no effect on the absorption of the free chromophore in an aqueous nonionic detergent solution. Reduction of retinal-RBP complexes with NaBH4 showed that the retinal aldehyde function was free and was not covalently linked to the protein. This was also shown by illuminating frozen solutions of retinal-RBP complexes with linearly polarized light (photoselection) and measuring the resulting linear dichroism spectrum. The linear dichroism spectrum of illuminated retinal-RBP complexes was similar to that of the corresponding free retinal isomer in aqueous digitonin solution. None of the retinal isomers-RBP and retinyl acetate-RBP complexes was bound to prealbumin (thyroxine-binding protein) at physiological ionic strength, whereas retinoic acid-RBP was bound to prealbumin under the same conditions as judged by gel filtration chromatography. On the other hand, the addition of prealbumin to retinal-RBP complexes at low ionic strength resulted in a shift to the blue of the absorption peak (8 nm with 9-cis-retinal-RBP). Increasing the ionic strength of a retinal-RBP and prealbumin solution resulted in a further shift to the blue (5 nm with 9-cis-retinal-RBP) and an increase in the area of the chromophore absorption band. Adding salt to a retinoic acid-RBP solution containing prealbumin resulted in a shift of 1 nm to the red and an increase in the absorption band. It was concluded from these experiments that although the retinals, retinoic acid, and retinyl acetate bind to retinol-binding protein at the same site as the retinol isomers, the binding of these various chromophores resulted in a somewhat altered conformation of the reconstituted retinol-binding protein. This in turn made it impossible for the retinals- and retinyl acetate-RBP complexes to bind to prealbumin and led to the various subtle differences in spectroscopic behavior of the various chromophore-RBP complexes upon changes in ionic strength and interaction with prealbumin.