Binding of Retinol-binding Protein Obtained from Human Urine with Vitamin A Derivatives and Terpenoids

Abstract

Retinol-binding protein (RBP) was purified from fresh urine of patients suffering from “ Itai-Itai ” disease. The purified preparation contained two types of apo-RBP (Apo I and II) in equal amounts as major components (about 85% of the total RBP). The corresponding two retinol-binding forms (Holo I and II) were present as minor components (about 15% of the total). Retinol-binding complex was produced by mixing an n-heptane solution of retinol with an aqueous solution of the purified apo-RBP preparation; the molar ratio of retinol to the protein in the reconstituted complex was 0.8, which is close to the equimolar ratio of retinol to RBP obtained from human plasma. Investigations of the binding of apo-RBP with various terpenoids revealed that an isoprene structure possessing conjugated double bonds is necessary for binding, which is strengthened by the presence of a β-ionone ring. Carboxylic acids tested were bound to apo-RBP with high affinity, possibly due to ionic binding of the carboxylic anion with the protein, supported by hydrophobic residues of the compounds. A carbonyl group promoted binding in the case of low molecular compounds, but was less effective in higher molecular compounds. A hydroxyl group was less effective for binding in small molecules, but was markedly effective in the case of retinol binding. The higher affinity of retinol with apo-RBP was probably due to the hydroxyl group, enhanced by the hydrophobic nature of the isoprene moiety. Retinol acetate and methyl retinoate did not bind to RBP at all, which appears to exclude the possibility that the hydrophobicity alone is responsible for the binding.