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Volume 272, Number 39, Issue of September 26, 1997 pp. 24455-24460
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and Partial Characterization of a Cellular Carotenoid-binding Protein from Ferret Liver

(Received for publication, January 24, 1997, and in revised form, May 28, 1997)

From the Department of Medicine, George Washington University, Washington, D. C. 20037 and the Lipid Research Laboratory, Department of Veterans Affairs Medical Center, Washington, D. C. 20422

A cellular carotenoid-binding protein was purified to homogeneity from -carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. [¹⁴C]-Carotene co-purified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to -carotene showed two classes of binding sites, a high affinity site with an apparent K_d of 56 × 10⁹ M and a low affinity site with a K_d of 32 × 10⁶ M. The B_max for -carotene binding to the high affinity site was 1 mol/mol, while that for the low affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in _max with minor peaks at 460 and 516 nm. Except for -carotene and cryptoxanthin, none of the model carotenoids or retinol competed with -carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted -ionone ring.

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