Vitamin A Transport in Rat Plasma: ISOLATION AND CHARACTERIZATION OF RETINOL-BINDING PROTEIN

Abstract

Studies were conducted to isolate and characterize rat serum retinol-binding protein (RBP), the specific transport protein for vitamin A in the rat. RBP was isolated from rat serum by a sequence of procedures which included: precipitation with ammonium sulfate between 30 and 50% saturation; chromatography on DEAE-Sephadex; gel filtration on Sephadex G-200 and G-100; and preparative polyacrylamide gel electrophoresis. These procedures resulted in RBP which had been purified approximately 2,300-fold, and which was completely pure by physical and by immunological criteria. Purified rat RBP has α1 mobility, a sedimentation constant (s20,w) of 2.06 S, and a molecular weight of approximately 20,000. The properties of rat RBP resemble those of human plasma RBP in many ways. The two proteins have nearly identical ultraviolet absorption spectra (peak maxima at 280 and 330 nm) and fluorescence emission and excitation spectra. The amino acid compositions of rat and human RBP are somewhat similar, both with a fairly high content of aromatic amino acids. A specific antirat RBP antiserum was prepared in a rabbit. There was no immunological cross-reactivity between rat and human RBP, indicating that the two proteins are immunologically completely distinct. In plasma, rat RBP circulates in the form of a protein-protein complex, with an apparent molecular weight of approximately 60,000 to 70,000. The protein (prealbumin-2) with which RBP interacts to form a complex has an electrophoretic mobility slightly greater than that of rat serum albumin. Purified prealbumin-2 has an apparent molecular weight of approximately 45,000 to 50,000. Prealbumin-2 may also represent a major transport protein for thyroid hormone in the rat.