Conformational Changes following Interaction between Retinol Isomers and Human Retinol-binding Protein and between the Retinol-binding Protein and Prealbumin

Abstract

Abstract Apo-retinol-binding protein (apo-RBP) from human plasma was found to be completely dissociated from prealbumin (thyroxine-binding protein) under conditions of physiological ionic strength in which native retinol-retinol-binding protein complex was tightly bound as judged by gel filtration chromatography. Addition of all-trans-retinol to apo-RBP resulted in (a) complete regeneration of the enhanced retinol fluorescence which is found in native retinol-binding protein (RBP), (b) 93 to 100% regeneration of the native absorption and circular dichroism spectra of native retinol-RPB, and (c) tight binding of the reconstituted retinol-RBP to prealbumin at physiological ionic strength. The binding between 9-, 11-, and 13-cis-retinol isomers and retinol-binding protein was compared to the binding of all-trans-retinol to the protein. All four retinol isomers were bound to retinol-binding protein in a molar ratio of 0.9 to 1.0. All four isomers were bound at the same site on the protein. The binding of all four retinols resulted in a 3- to 5-mn red shift of the chromophore absorption peak, and it led to some 10-fold enhancement of the fluorescence quantum yield. All four isomers showed an extrinsic Cotton effect of the chromophore absorption band. The rotatory strength of the all-trans-retinol-RBP was 3 times greater than that of the other retinol-RBP complexes. All the retinol isomers-RBP complexes showed small but variable changes in the chromophore absorption and circular dichroism bands upon increase in ionic strength. All the retinol-RBP complexes were bound to prealbumin (thyroxine-binding protein) at physiological ionic strength, and this binding resulted in a 10% increase of the chromophore absorption band and a variable effect on the circular dichroism band at 330 nm. These changes in the absorption occurred in less than 30 s after the addition of salt. Addition of salt to prealbumin alone had no effect on the absorption band between 240 and 300 nm. The binding between retinol isomers-RBP and prealbumin resulted in a nonlinear addition of the circular dichroism spectra of retinol-binding protein and prealbumin in the 250 to 300 nm range. Linear addition of the circular dichroism spectra was observed when the two proteins were mixed under conditions in which they did not form a stable complex. Such conditions were found to be low ionic strength buffers, 8 m urea, and 6 m guanidine HCl. Changes in ionic strength or addition of urea or guanidine HCl to the individual proteins did not lead to any significant changes in the circular dichroism spectra. Removal of the urea or guanidine HCl by dialysis led to the formation of a complex between retinol-RBP and prealbumin, as judged by the reappearance of the changes (nonlinear addition) in the circular dichroism spectra. It was concluded from this series of experiments that removal of retinol from retinol-binding protein results in a conformational change such as to make apo-RBP unable to bind to prealbumin. The binding of retinol-RBP to prealbumin results in conformational changes in either the retinol-binding protein, the prealbumin, or both. The native retinol-RBP which is isolated from human plasma is the all-trans isomer.