Studies on Two Physiological Forms of the Human Retinol-binding Protein Differing in Vitamin A and Arginine Content

Abstract

Abstract The human retinol-binding protein (RBP) is shown to exist in two main physiological forms each of which displays electrophoretic heterogeneity. Only one of the two RBP components contains vitamin A. The retinol-containing RBP species is under physiological conditions firmly bound to thyroxine-binding prealbumin. The protein complex thereby formed is the actual vitamin A carrier normally encountered in plasma. The vitamin A-free form of RBP is unable to bind prealbumin. The difference in the affinity of the two RBP species for prealbumin made it possible to separate them by chromatography on a Sepharose gel to which prealbumin had been covalently attached. Differences in conformation between the two RBP components were established by physical-chemical, chemical, and immunological techniques. On polyacrylamide gel electrophoresis both species of RBP exhibited multiple bands. The studies revealed that the variations in electrophoretic mobility could be ascribed to differences in the content of vitamin A, in the number of amide groups, and in the sequence of the COOH-terminal amino acid residues. Hence, it was found that the vitamin A-containing form of RBP had an additional residue of arginine compared to the form lacking the vitamin. This result, obtained by amino acid analysis, was confirmed by peptide mapping, which revealed an arginine-stainable spot present only in the vitamin A-containing species. The COOH-terminal amino acid sequences in the two RBP species were -Lys-Arg-OH and -Lys-OH, respectively. The loss of 1 amino acid residue from the vitamin A-free species obviously causes a conformational change which is not compatible with the binding to prealbumin. Vitamin A per se is not a prerequisite for the binding of RBP to prealbumin since it was shown that RBP from which the vitamin had been extracted retained its affinity for prealbumin. The data obtained in this study strongly suggest that the species devoid of both vitamin A and the COOH-terminal arginine is a catabolite of RBP.