Cellular retinol- and retinoic acid-binding proteins of bovine retina. Purification and properties.

A three-step purification has been developed for the cellular retinoland cellular retinoic acid-binding proteins from extracts of bovine retina. The two binding proteins elute together in Step 1 (gel filtration) and separate in Step 2 (DEAE-cellulose chromatography), and each is purified to apparent homogeneity in the third step (calcium phosphate gel chromatography). The retinoland retinoic acid-binding proteins are both acidic, of similar molecular weight (16,600 + 200 and 16,300 + 300, respectively), similar but not identical in amino acid composition, and form one-to-one complexes with their respective ligands. A comparison of the amino acid compositions of the retinoland retinoic acid-binding proteins with that of the serum retinolbinding protein eliminates the latter as a precursor for either of the two cellular proteins. Interaction of retinol and the cellular retinol-binding protein results in the appearance of fine structure in the absorption spectrum of the ligand and a red shift in the observed absorption maximum. The ratio of absorbance at 350 nm to that at 280 nm in isolated retinolbinding protein is 1.65. Retinoic acid bound to its binding protein produces an absorption spectrum similar to that of the retinoate anion (X,,,,, = 342 nm). The interaction of retinoic acid with its binding protein induces fluorescence of the ligand. Fluorometric titration of aporetinoic acid-binding protein with retinoic acid suggested that 1 mol of retinoic acid is bound/m01 of binding protein.