Characterization of a Novel α-Tocopherol-Binding Protein from Bovine Heart Cytosol

We previously reported the identification of a new α-tocopherol-binding protein (∼ 15 kDa) in the cytosol of rat liver and heart and in rabbit heart (A, K, Dutta-Roy et al., J, Nutr, Biochem. 5, 562-570, 1994). This protein specifically binds α-tocopherol and enhances its transfer between separate membranes, In the present paper we have purified and characterized the α-tocopherol-binding protein from bovine heart cytosol and compared its various structural and functional properties with the similar size (∼ 15 kDa) cytosolic fatty acid-binding protein of this tissue. α-Tocopherol-binding protein was purified to electrophoretic homogeneity from bovine heart cytosol by a procedure involving precipitation with 70% ammonium sulfate, followed sequentially by gel filtration chromatography and chromatofocusing, The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 16 kDa, Isoelectric focusing of the purified protein showed that the pI value is around 4.5. The binding of α-tocopherol to the purified protein was rapid, reversible, and saturable. The α-tocopherol-binding protein did not bind oleate as assessed by direct radiolabeled fatty acid binding and fluorescence enhancement assay, Amino acid analysis showed the presence of a large number of Ala, Gly, Ser, Lys, and Pro residues and a lesser number of aromatic residues in this protein. Anti-bovine heart fatty acid-binding protein antibody did not recognize the α-tocopherol-binding protein in the Western blot. The Western blot, ligand affinity, molecular size, and amino acid analysis data suggest that the α-tocopherol-binding protein is different from the cytosolic fatty acid-binding protein and that it may be involved in intracellular transport and metabolism of α-tocopherol.