Influence of Phosphatidylcholine and Calcium on Self-Association and Bile Salt Mixed Micellar Binding of the Natural Bile Pigment, Bilirubin Ditaurate.
Recently [Neubrand, M. W., et al. (2015) Biochemistry 54, 1542-1557], we determined a concentration-dependent monomer-dimer-tetramer equilibrium in aqueous bilirubin ditaurate (BDT) solutions and explored the nature of high-affinity binding of BDT monomers with monomers and micelles of the common taurine-conjugated bile salts (BS). We now investigate, employing complementary physicochemical methods, including fluorescence emission spectrophotometry and quasi-elastic light scattering spectroscopy, the influence of phosphatidylcholine (PC), the predominant phospholipid of bile and calcium, the major divalent biliary cation, on these self-interactions and heterointeractions. We have used short-chain, lyso and long-chain PC species as models and contrasted our results with those of parallel studies employing unconjugated bilirubin (UCB) as the fully charged dianion. Both bile pigments interacted with the zwitterionic headgroup of short-chain lecithins, forming water-soluble (BDT) and insoluble ion-pair complexes (UCB), respectively. Upon micelle formation, BDT monomers apparently remained at the headgroup mantle of short-chain PCs, but the ion pairs with UCB became internalized within the micelle's hydrophobic core. BDT interacted with the headgroups of unilamellar egg yolk (EY) PC vesicles; however, with the simultaneous addition of CaCl2, a reversible aggregation took place, but not vesicle fusion. With mixed EYPC/BS micelles, BDT became bound to the hydrophilic surface (as with simple BS micelles), and in turn, both BDT and BS bound calcium, but not other divalent cations. The calcium complexation of BDT and BS was enhanced strongly with increases in micellar EYPC, suggesting calcium-mediated cross-bridging of hydrophilic headgroups at the micelle's surface. Therefore, the physicochemical binding of BDT to BS in an artificial bile medium is influenced not only by BS species and concentration but also by long-chain PCs and calcium ions that exert a specific rather than a counterion effect. This work should serve as a physicochemical template for studies with other conjugated bilirubins, including bilirubin diglucuronoside (BDG), the principal bilirubin conjugate (cBR) in human bile.
Department of Medicine, Harvard Medical School, and Division of Gastroenterology, Brigham and Women's Hospital and Harvard Digestive Disease Center , Boston, Massachusetts 02115, United States.|Department of Biochemistry, University of New Hampshire , Durham, New Hampshire 03824, United States.
Michael W
MW
Neubrand
Department of Medicine, Harvard Medical School, and Division of Gastroenterology, Brigham and Women's Hospital and Harvard Digestive Disease Center , Boston, Massachusetts 02115, United States.|Department of Biochemistry, University of New Hampshire , Durham, New Hampshire 03824, United States.
Martin C
MC
Carey
Department of Medicine, Harvard Medical School, and Division of Gastroenterology, Brigham and Women's Hospital and Harvard Digestive Disease Center , Boston, Massachusetts 02115, United States.|Department of Biochemistry, University of New Hampshire , Durham, New Hampshire 03824, United States.
Thomas M
TM
Laue
D016880:Anisotropy; D001647:Bile Acids and Salts / Q000737:chemistry*; D001663:Bilirubin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001704:Biopolymers; D002122:Calcium Chloride / Q000737:chemistry*; D019281:Dimerization; D008823:Micelles; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000737:chemistry*; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013654:Taurine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D053835:Unilamellar Liposomes
D001647:Bile Acids and Salts; D001704:Biopolymers; D008823:Micelles; D010713:Phosphatidylcholines; D012996:Solutions; D053835:Unilamellar Liposomes; D013654:Taurine; C054146:bilirubin ditaurine; D002122:Calcium Chloride; D001663:Bilirubin