Properties of the Chromophore Binding Site of Retinolbinding Protein from Human Plasma

Abstract

Abstract Apo-retinol-binding protein (apo-RBP) from human plasma was found to bind all-trans-3-dehydroretinol and to protect the chromophore from degradation. The all-trans-3-dehydroretinol-RBP complex, in turn, binds tightly to prealbumin at physiological ionic strength as judged by gel filtration chromatography. All-trans-3-dehydroretinal also formed a complex with RBP but this complex did not bind to prealbumin. Both the all-trans-3-dehydroretinol and all-trans-3-dehydroretinal-RBP complexes showed induced optical activity of the chromophore absorption spectrum. In marked contrast to the retinol- and retinal-RBP complexes the sign of the long wavelength circular dichroism band of the all-trans-3-dehydro chromophore-RBP complexes was reversed. The chromophoric binding site of apo-RBP was probed for binding with hydrophobic compounds such as β-ionone, crocetin, and 1-phenyldecane. All of these compounds did not bind to apo-RBP, as was judged by the availability of the binding site to the native chromophore-retinol. This suggests that the retinyl moiety is necessary for binding. The capacity of the chromophoric binding site to bind to various retinyl derivatives was tested with several retinyl-Schiff bases. Retinylideneaniline did not bind to apo-RBP, but when the phenyl group was displaced by adding a C4 chain between the retinyl and the phenyl moieties as in retinylidene-4-phenylbutylamine, binding to RBP occurred. Aliphatic retinyl-Schiff bases shorter than and including retinylidenehexylamine bind to apo-RBP. With retinylidenedecylamine only 30 to 40% of the binding site was occupied. Retinylidene-1-tetradecylamine did not bind at all to apo-RBP. Similarly, retinyl palmitate did not bind to apo-RBP. The fact that liver retinol is stored as retinyl palmitate suggests that the palmitate group must be enzymatically hydrolyzed before the retinol can bind to apo-RBP, so as to be released from the liver stores. This enzymatic step makes it possible to control the rate of retinol release from the liver and its level in the plasma.