Isolation and Properties of a Human Retinol-transporting Protein

Abstract

A human retinol-transporting protein has been isolated from the urine of patients with tubular proteinuria. The protein separated into four fractions in the last two purification steps (sulfoethyl Sephadex chromatography and DEAE-Sephadex chromatography). The yield of purified retinol-binding protein (RBP) was 33 to 41% of the amount in concentrates of urinary proteins. A substance with an absorbance maximum at 330 nm was extracted from the protein with heptane and identified as retinol. Close to 1 mole of retinol per mole of protein was present in the fraction with the highest vitamin A content. The four isolated fractions of RBP gave reactions of identity on Ouchterlony immunodiffusion and a total of four zones on polyacrylamide gel electrophoresis. Isoelectric focusing also revealed four components with isoelectric points in the pH range of 4.4 to 4.8. Ultracentrifugations indicated that the two main protein fractions were homogeneous with a molecular weight of 21,400. Amino acid analyses suggested that these two fractions differed by 1 residue of arginine. RBP was free from neutral sugar and hexosamine. Immunochemical determinations of RBP in normal biological fluids gave the following average values: serum, 46 mg per liter; urine, 0.11 mg per 24-hour volume; and cerebrospinal fluid, 0.35 mg per liter. Urine from patients with tubular proteinuria contained up to 150 mg of RBP per 24-hour volume.