Abstract
It has been proposed that the neurotoxicity observed in severely jaundiced infants results from the binding of unconjugated bilirubin to nerve cell membranes. However, despite potentially important clinical ramifications, there remains significant controversy regarding the physical nature of bilirubin-membrane interactions. We used the technique of parallax analysis of fluorescence quenching (Chattopadhyay, A., and E. London. 1987. Biochemistry. 26: 39–45) to measure the depth of penetration of bilirubin in model phospholipid bilayers. The localization of unconjugated bilirubin and ditaurobilirubin within small unilamellar vesicles composed of dioleoylphosphatidylcholine was determined through an analysis of the quenching of bilirubin fluorescence by spin-labeled phospholipids, and by bilirubin-mediated quenching of a series of anthroyloxy fatty acid probes at various depths within the membrane bilayer. Findings were further verified with potassium iodide as an aqueous quencher. Our results indicate that, at pH 10, unconjugated bilirubin localizes approximately 20 Å from the bilayer center, in the region of the polar head groups. Further analyses suggest a modest influence of pH, membrane cholesterol content, and vesicle diameter on the bilirubin penetration depth. Taken together, these data support that, under physiologic conditions, bilirubin localizes to the polar region of phospholipid bilayers, near the membrane-water interface. —Zucker, S. D., W. Goessling, E. J. Bootle, and C. Sterritt. Localization of bilirubin in phospholipid bilayers by parallax analysis of fluorescence quenching.
Highlights
It has been proposed that the neurotoxicity observed in severely jaundiced infants results from the binding of unconjugated bilirubin to nerve cell membranes
In an attempt to resolve these conflicting data, we used the method of parallax analysis of fluorescence quenching, Abbreviations: 2, 6, 9, or 12-AS, 2, 6, 9, or 12-(9-anthroyloxy)stearic acid; BDT, ditaurobilirubin; DOPC, 1,2-dioleoyl-sn-glycero-3phosphocholine; Large unilamellar vesicles (LUV), large unilamellar vesicles; 5- or 12-SLPC, 1-palmitoyl-2-(5- or 12-doxyl)stearoyl-sn-glycero-3phosphocholine; Small unilamellar vesicles (SUV), small unilamellar vesicles
Phosphatidylcholine labeled with a doxyl moiety at either the 5- or 12-position was incorporated into DOPC vesicles at molar ratios ranging from 0% to 75%
Summary
Bilirubin (unconjugated bilirubin) and ditaurobilirubin (bilirubin conjugate-ditaurate·2Na; BDT) were obtained from Porphyrin Products (Logan, UT). Cholesterol and/or fluorescently labeled probes were incorporated into vesicles by adding the lipids, at specified molar ratios, to the chloroform solution of DOPC before desiccation. DOPC vesicles was achieved by examining the quenching of probe fluorescence intensity by a series of spin-labeled phospholipids located at defined depths within the bilayer. Fluorescence readings were obtained by briefly opening the excitation slit and recording the average intensity over 3 s, thereby minimizing the exposure of the sample to incident light. This approach to sample handling resulted in trivial (р2%) loss of pigment to photo-oxidation, as assessed by absorption spectroscopy. Because accurate localization of spin-labeled phosphatidylcholine probes within DOPC bilayers previously has been established [22], the average distance from the center of the bilayer to the bilirubin molecule (zcB) can be calculated by equation 1 [22], zcB ϭ
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