The Interaction of Human Plasma Retinol-binding Protein with Prealbumin

Abstract

Abstract The interaction of human plasma retinol-binding protein with plasma prealbumin was studied by the techniques of velocity ultracentrifugation and polarization of retinol fluorescence. In the first method the unbound fraction of retinol-binding protein, which sediments more slowly than its complexes with prealbumin, was measured by its absorption. A stoichiometry of retinol-binding protein to prealbumin, 4:1 was found. The polarization of fluorescence of retinol increases when retinol-binding protein combines with prealbumin; the relaxation time of retinol-binding protein was 6.6 x 10-8 s at 25° and increased to 28 x 10-8 s for the complex. The polarization data indicate that there are four independent binding sites with an apparent association constant of approximately 1.2 x 106 m-1 at 25° and pH 7.4. The affinity was maximal near pH 7.4 and decreased gradually at lower and higher pH values. Between pH 6.0 and 9.4, the extent of interaction between retinol-binding protein and prealbumin was dependent on ionic strength. In this pH range almost no binding occurred without added salt and the affinity increased rapidly with increasing KCl concentration to 0.1 m. At pH values below 6 the interaction was less dependent on ionic strength and became independent at pH 4.2.