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.

Highlights

  • 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

  • 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

  • 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

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Summary

SUMMARY

The reconstitution of analogs of the native chromophore retinol (vitamin A) with apo-retinol-binding protein 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-. It was concluded from these experiments that the retinals, retinoic acid, and retinyl acetate bind to retinolbinding protein at the same site as the retinol isomers, the binding of these various chromophores resulted in a somewhat altered conformation of the reconstituted retinolbinding 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. The retinol isomer-RBP complexes show a hyperchromic effect of the chromophore absorption band when salt is added to a low ionic strength buffer in the presence of prealbumin. We report on the interaction of these complexes with prealbumin

PROCEDURE
I-cis retinal-RBP
Findings
I-cis retinal
Full Text
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