A new ligand for an old lipocalin: induced circular dichroism spectra reveal binding of bilirubin to bovine β-lactoglobulin

Abstract

This study reports that bilirubin–bovine β-lactoglobulin (BLG) complexes exhibit very characteristic induced circular dichroism (CD) spectra in the visible absorption region. Due to intramolecular chiral exciton coupling between the dipyrrinone chromophores, the long-wavelength negative and short-wavelength positive CD bands clearly prove that a single bilirubin molecule binds to BLG in a left-handed helical conformation (in pH 7.4 phosphate buffer Δ ϵ min is −54 M −1 cm −1 at 467 nm and Δ ϵ max is +48.5 M −1 cm −1 at 412 nm). The very low aqueous solubility and strong tendency of bilirubin molecules to aggregate around pH 7.4 meant that much more intense CD bands were measured at alkaline pH values owing to the increasing solubility of the ligand. Vanishing CD activity obtained upon titration of the complex with palmitic acid known to bind in the hydrophobic cavity of BLG indicates bilirubin to be bound at the open end mouth of the β-barrel. Reversible changes of the induced CD spectrum due to acidic pH shift of the sample solution lead to the same conclusion.