SELF-ASSOCIATION OF UNCONJUGATED BILIRUBIN-IX-ALPHA IN AQUEOUS-SOLUTION AT PH10.0 AND PHYSICAL-CHEMICAL INTERACTIONS WITH BILE-SALT MONOMERS AND MICELLES

Spectrophotometric measurements of bilirubin-IXα in water and aqueous/organic solvent mixtures at pH 10.0 as a function of bilirubin-IXα concentration (approx. 0.6-400 μM) are consistent with the formation of dimers (Kp=1.5 μM) aqueous solution and further self-aggregation to multimers at higher concentrations. Added urea (to 10 M) and increases in temperature (to 62(o)C) obliterate the dimer-multimer transition at 10 μM, but added NaC1 (to 0.30 M) promotes strong aggregation of dimers over a narrow concentration range, suggesting a 'micellization' phenomenon. Concentrations of dioxan or ethanol greater than 60% (v/v) in water were required to obtain the absorption spectrum of bilirubin-IXα manomers, suggesting that both hydrophobic and electrostatic (π-orbital) interactions are involved in stabilizing the dimeric state in water. Micellar concentrations of sodium dodecyl sulphate induced spectrophotometric shifts in the dimer absorption spectrum of bilirubin-IXα consistent with progressive partitioning of bilirubin-IXα monomers into a relatively non-polar region of the micelles and allowed a deduction of the apparent critical micellar concentration that closely approximated the literature values. The pattern of bilirubin-IXα association with bile salts is complex, since the absorption spectrum shifts hypsochromically below and bathochromically above the critical micellar concentration of the bile salts. Consistent with these observations, bilirubin IXα appears to bind to the polar face of bile salt monomers and to the polar perimeter of small bile salt micelles. At higher bile salt concentrations some bilirubin-IXα may exhibit similar physical chemical properties in bile, in that dimers, highly aggregated multimers and the bile salt-associated monomers may co-exist.