Vitamin A transport in chicken plasma: isolation and characterization of retinol-binding protein (RBP), prealbumin (PA), and RBP--PA complex.

Vitamin A-transporting protein in chicken plasma was purified by column chromatography on DEAE-Sephadex and Sephadex G-100; the protein formed a complex of retinol-binding protein (RBP) with prealbumin (PA). The molecular weight of the 1:1 molar complex was estimated to be 76,000 by gel filtration, and the sedimentation coefficient (S20,W) was found to be 5.2 S. RBP and PA were dissociated from the purified complex by means of CM-Sephadex column chromatography. Purified RBP contained 1 mole of vitamin A bound per mole of RBP. The molecular weight of RBP was determined to be 20,000 by gel filtration on Sephadex G-75, 19,000 by SDS-disc gel electrophoresis, and 20,500 by sedimentation equilibrium analysis. The S20,W was calculated to be 2.0 S. The molecular weight of PA was determined to be 56,000 by gel filtration, 52,000 by sedimentation equilibrium analysis, and 13,000 by SDS-disc gel electrophoresis. The S20,W was calculated to be 3.9 S. From these findings it was concluded that PA consists of four subunits, each with a molecular weight of approximately 13,000. Peptide mapping experiments suggested that the subunits were identical. No carbohydrates were detected in either RBP or PA. Chicken RBP and PA were immunologically distinct from those of human and rat. It is well established that vitamin A is transported bound to a specific plasma protein, retinol-binding protein (RBP), in both man (1,2) and rat (3). Purified human and rat plasma RBP have a single binding site for one molecule of retinol, alpha mobility on disc gel electrophoresis, and a molecular weight of approximately 20,000. In both species, RBP forms a tight complex with plasma prealbumin (PA) and normally circulates as a 1:1 molar protein-protein complex with PA (1-5). Despite these similarities, no immunological cross-reactivity between human and rat RBP has been observed (3,6). The present study was undertaken to explore whether or not a similar transport system for vitamin A exists in the chicken, a nonmammalian vertebrate. During the course of this study, Mokady and Tal (7) reported the isolation of RBP from chicken plasma and some physicochemical properties, e.g., a molecular weight of about 19,000. On the other hand, Muto, Smith, and Goodman (6) had already observed that the molecular weight of vitamin A-containing protein in fresh chicken plasma is approximately 60,000-80,000, as determined by gel filtration. However, no convincing information is available regarding an entire system of vitamin A transport in chicken plasma. We now describe procedures for the isolation of the RBA-PA complex of chicken plasma and the dissociation into the component proteins, RBP and PA. We also describe in detail the physicochemical properties of the individual proteins. It is also clearly demonstrated that chicken RBP and PA are immunologically distinct and different from the respective proteins in man and rat. Moreover, purified chicken PA appears to be a tetramer of four identical subunits and is thus similar to human and rat PA.