Isolation of the human retinol binding protein by affinity chromatography.

Abstract

The human retinol binding protein, which in plasma is bound to thyroxine binding prealbumin, has been isolated in free form from urine of patients with tubular proteinuria and from normal serum. The isolation procedure for urinary retionl binding protein involved gel chromatography and affinity chromatography on a column of prealbumin coupled to Sepharose, whereas the isolation of serum retinol binding protein required three fractionation steps; DEAE‐Sephadex chromatography, and affinity chromatography.The retinol binding protein from urine and serum was obtained in a yield of 31% and 42% respectively. The relatively low yield of the urinary protein was explained by the fact that two forms of retinol binding protein were present in the urine, one of which did not bind to prealbumin. The purity of the retinol binding protein isolated from urine and serum was established by the following criteria: immunoelectrophoresis, polyacrylamide gel electrophoresis, amino acid analyses, and sedimentation equilibrium ultracentrifugations.The use of affinity chromatography offers a simple and rapid procedure for the isolation of moderate quantities of highly purified retinol binding protein from urine and serum.