β‐Lactoglobulin binds retinol and protoporphyrin IX at two different binding sites

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Abstract
Measurement of tryptophan fluorescence quenching and the excitation energy transfer from tryptophanyl residues to the bound ligand indicates that β-lactoglobulin binds tightly to hemin and protoporphyrin IX in a ligand-to-protein stoichiometric ratio. The apparent dissociation constants of hemin-β-lactoglobulin and protoporphyrin IX-β-lactoglobulin complexes are 2.5 × 10−7 M and 4 × 10−7 M, respectively. The addition of β-lactoglobulin (final concentration = 10 μM, phosphate buffer 50 mM, pH 7.1) to the solution containing retinol and protoporphyrin IX triggers an energy transfer between β-lactoglobulin tryptophan and protoporphyrin IX as well as between retinol and protoporphyrin IX. The efficiency of energy transfer depends on the distance between the donor (retinol) and the acceptor (protoporphyrin IX). Using the Förster theory, a retinol-protoporphyrin IX distance of 25 Å was calculated. These results indicate that retinol and protoporphyrin IX are bound to the β-lactoglobulin monomer at two different sites.