Self-Assembly of Aqueous Bilirubin Ditaurate, a Natural Conjugated Bile Pigment, to Contraposing Enantiomeric Dimers and M(−) and P(+) Tetramers and Their Selective Hydrophilic Disaggregation by Monomers and Micelles of Bile Salts

The solution behavior of bilirubin ditaurate (BDT), the first naturally occurring conjugated bile pigment to be physically and chemically characterized, was assessed in aqueous solution and in monomeric and micellar solutions of common taurine-conjugated bile salts (BS). Analytical ultracentrifugation revealed that BDT self-associates in monomer–dimer equilibria between 1 and 500 μM, forming limiting tetramers at low millimolar concentrations. Self-association was enthalpically driven with ΔG values of ≈5 kcal/mol, suggesting strong hydrophobic interactions. Added NaCl and decreases in temperature shifted the oligomerization to lower BDT concentrations. On the basis of circular dichroism spectra and the limiting size of the self-aggregates, we infer that the tetramers are composed of 2P(+) and 2M(−) enantiomeric BDT pairs in “ridge-tile” conformations interacting in a “double-bookend” structure. With added monomeric BS, blue shifts in the UV–vis spectra and tight isosbestic points revealed that BDT/BS heterodimers form, followed by BDT “decorating” BS micelles mostly via hydrophilic interactions. Conformational enantiomerism, fluorescence intensities, and anisotropy, as well as resistance of the hybrid particles to disaggregation in 6 M urea, suggested that two or three hydrogen-bonding sites bound BDT monomers to the hydroxyl groups of BS, possibly via pyrrole−π-orbital–OH interactions. BDT stabilized these interactions by enveloping the BS in its “ridge-tile” pincers with variable strain that maximized van der Waals interactions. Possibly because the BDT molecule becomes highly strained with BS subtending a 7β-hydroxyl group, BDT became totally resistant to oxidation in air. This work predicts that, because of BS dissolution of the BDT self-aggregates, BS/bilirubin hybrid particles, which are stabilized hydrophilically, are likely to be the dominant mode of transport for all conjugated bilirubins in bile