Solubilization of bilirubin by cholate micelles. Spectroscopic and gel permeation studies

The interactions of bilirubin with bile salts were studied by fluorescence, CD, and 1H NMR methods. Enhancement of bilirubin fluorescence and induction of optical activity in bilirubin in the presence of cholate has been obsd. Fluorescence enhancement is pronounced above the crit. micelle concn., whereas induced CD bands are detectable even in the premicellar region. Dehydrocholate and deoxycholate did not increase fluorescence, but did induce CD bands for bilirubin. Gel-permeation chromatog. on Sephadex G-50 yielded a single bilirubin-cholate species at alk. pH, whereas 2 species were obtained at neutral pH. 1H NMR and CD spectral characterizations of these complexes are reported. A 4:1 cholate-bilirubin mixt. was analyzed by difference NOE spectroscopy. Observation of strong, neg. NOE, both intermol. and intramol., suggests that specific Me groups on bilirubin and cholate are proximal in the mixed micelle

A proton-NMR study of bilirubin IX\alpha solubilization by cholate micelles: application of nuclear Overhauser effects.

The solubilization of bilirubin IX\alpha (I) in aq. soln. by Na cholate micelles was examd. by 270-MHz 1H NMR spectroscopy. The incorporation of I into the micelles was accompanied by specific shifts of I vinyl and bridgehead protons and the C18 and C19 Me groups of the steroid. The obsd. chem. shifts showed a monotonic concn. dependence suggesting that changes in aggregation size are continuous. Nuclear Overhauser effects (NOE) were shown to be a useful probe or micellization. A 4:1 cholate (II)/I mixt. was investigated by difference NOE spectroscopy. The observation of intermol. NOE between peripheral protons of I and II were diagnostic of spatially proximate groups. Inter-II NOE increased in magnitude upon I incorporation, suggesting closer packing of steroid mols. on solubilization of the pigment. Intramol. NOE obsd. for solubilized I were consistent with a compact intramolecularly H-bonded conformation