MECHANISM OF THE SPONTANEOUS TRANSFER OF UNCONJUGATED BILIRUBIN BETWEEN SMALL UNILAMELLAR PHOSPHATIDYLCHOLINE VESICLES

Unconjugated bilirubin (bilirubin-IX-alpha), the hydrophobic end produ ct of heme degradation, is esterified in the hepatocyte endoplasmic re ticulum to water-soluble conjugates prior to excretion in bile. To cha racterize the process of intracellular bilirubin transport, the kineti c and thermodynamic activation parameters for the spontaneous transfer of bilirubin between small unilamellar egg lecithin vesicles were det ermined. Bilirubin-IX-alpha was added to donor vesicles labeled with t he fluorescent phospholipid probe, fonyl)dipalmitoyl-L-alpha-phosphati dylethanolamine (dansyl-PE). When bound to the donor vesicles, bilirub in quenches the dansyl probe fluorescence through resonance energy tra nsfer. The movement of bilirubin from dansyl-labeled donor vesicles to unlabeled acceptor vesicles was monitored directly by the reemergence of dansyl fluorescence over time. Vesicle fusion and intervesicle tra nsfer of the dansyl-PE probe were excluded by quasielastic light scatt ering and fluorescence resonance energy transfer studies. Stopped-flow analysis demonstrated that the transfer of bilirubin was described by a single-exponential function with a mean half-time of 2.0 +/- 0.1 ms (+/- SD) at 37-degrees-C. The rate of bilirubin transfer was independ ent of acceptor vesicle concentration and decreased with increasing bu ffer ionic strength, indicating that intermembrane transfer occurred v ia aqueous diffusion, rather than vesicle collisions. The free energy of activation (DELTA-G) for the dissociation of bilirubin from donor v esicles was 14.2 kcal-mol-1. These studies suggest that bilirubin is a ssociated with phospholipid bilayers at the membrane-water interface. We postulate that the movement of unconjugated bilirubin between intra cellular membranes occurs via spontaneous transfer through the aqueous phase.