Studies on the Protein-Protein and Protein-Ligand Interactions Involved in Retinol Transport in Plasma

Abstract

Abstract Studies were conducted to examine the chemical and physical properties of human plasma retinol-binding protein (RBP) and prealbumin (PA), and the factors affecting their interaction with each other and with the ligands retinol and thyroxine, respectively. The effects of changes in temperature, pH, and urea concentration and the effects of reduction and alkylation of disulfide bonds and of iodination were assessed. Analytical ultracentrifuge studies established the formation of a 1:1 molar complex between the two proteins. The estimated molecular weight of the complex (approximately 70,000) was almost exactly the sum of the molecular weights of the individual proteins (PA, approximately 49,000 and RBP, 21,000). The complex was stable in the temperature range 4–42.5° and in the pH range 5.8 to 7.5. Considerable dissociation of the complex occurred as the pH was raised from 8.6 to 10.3. Addition of 6 m urea completely disrupted the RBP-PA complex, markedly reduced the affinity of PA for thyroxine, but did not interfere with the association of retinol with RBP. Only one of the two disulfide bonds of PA was readily accessible to reduction and alkylation. Reduction of this one disulfide bond did not affect the binding of either RBP or thyroxine to PA. In contrast, both disulfide bonds of RBP were highly resistant to reduction. When the disulfide bonds of RBP were reduced and alkylated, however, the retinol-RBP complex was disrupted, and retinol was completely lost from the protein. Iodination of RBP up to the level of 4.3 atoms of iodine per molecule selectively interfered with the interaction of RBP with PA, without disrupting the retinol-RBP complex. These studies demonstrate that it is possible to affect selectively the different interactions involved in the RBP-PA system responsible for retinol transport in plasma.