Molecular and Cellular Biochemistry

, Volume 277, Issue 1–2, pp 101–107

Interaction of bilirubin with sealed and human serum albumin-entrapped sealed membranes

  • Huma Rashid
  • Mohammad Mushahid Khan
  • Saad Tayyab


In order to study the mechanism of entry and localization of bilirubin (BR) into cell membrane, binding of BR to sealed and human serum albumin (HSA)-entrapped sealed membranes was studied by CD spectroscopy. An induced bisignate CD cotton effects (CDCEs) of BR-bound sealed membranes were observed with maxima at 515 nm and minima at 470 nm with a shoulder at 430 nm. BR-bound HSA-entrapped sealed membranes produced CD spectra with additional positive peaks at 450 and 475 nm and negative troughs at 390 and 415 nm. The induced CDCEs of BR-bound sealed membranes and BR-bound HSA-entrapped sealed membranes were perturbed by the addition of drugs (ceftriaxone and sodium salicylate) with the effect of ceftriaxone being more pronounced. Drugs’ being the displacer of BR from albumin, their incorporation in the incubation mixture was paralleled by reduction in CDCEs. Taken together, these results suggest that BR can traverse the membrane bilayer towards the inner surface instead of remaining intercalated in the exterior half of the bilayer.


bilirubin-albumin interaction bilirubin binding CD spectroscopy drug-induced bilirubin displacement human erythrocyte membrane 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Hansen TWR, Bratlid D: Bilirubin and brain toxicity. Acta Paediatr Scand 75: 513–522, 1986PubMedGoogle Scholar
  2. 2.
    Ostrow JD, Pascolo L, Shapiro SM, Tiribelli C: New concepts in bilirubin encephalopathy. Eur J Clin Invest 33: 988–997, 2003CrossRefPubMedGoogle Scholar
  3. 3.
    Pascolo L, Del Vecchio S, Koehler RK, Bayon JE, Webster CC, Mukerjee P, Ostrow JD, Tiribelli C: Albumin binding of unconjugated [3H] bilirubin and its uptake by rat liver basolateral plasma membrane vesicles. Biochem J 316: 999–1004, 1996PubMedGoogle Scholar
  4. 4.
    Sato H, Aono S, Semba R, Kashiwamata S: Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes. Biochem J 248: 21–26, 1987PubMedGoogle Scholar
  5. 5.
    Vazquez J, Garcia-Calvo M, Valdivieso F, Mayor F, Mayor F Jr: Interaction of bilirubin with synaptosomal plasma membrane. J Biol Chem 263: 1255–1265, 1988PubMedGoogle Scholar
  6. 6.
    Serrano MA, Bayon JE, Pascolo L, Tiribelli C, Ostrow JD, Gonzalez-Gallego J, Marin JJ: Evidence for carrier-mediated transport of unconjugated bilirubin across plasma membrane vesicles from human placental trophoblast. Placenta 23: 527–535, 2002CrossRefPubMedGoogle Scholar
  7. 7.
    Leonard M, Noy N, Zakim D: The interaction of bilirubin with model and biological membranes. J Biol Chem 264: 5648–5652, 1989PubMedGoogle Scholar
  8. 8.
    Tayyab S, Ali MK: Binding of bilirubin to erythrocytes from different mammalian species. Comp Biochem Physiol 111: 507–509, 1995CrossRefGoogle Scholar
  9. 9.
    Tayyab S, Ali MK: Binding of bilirubin to mammalian erythrocytes. Comp Biochem Physiol 118: 97–103, 1997CrossRefGoogle Scholar
  10. 10.
    Sato H, Kashiwamata S: Interaction of bilirubin with human erythrocyte membranes. Biochem J 210: 489–496, 1983PubMedGoogle Scholar
  11. 11.
    Hayward D, Schiff D, Fedunec S, Chan G, Davis PJ, Poznansky MJ: Bilirubin diffusion through lipid membranes. Biochim Biophys Acta 860: 149–153, 1986PubMedGoogle Scholar
  12. 12.
    Noy N, Leonard M, Zakim D: The kinetics of interactions of bilirubin with lipid bilayers and with serum albumin. Biophys Chem 42: 177–188, 1992CrossRefPubMedGoogle Scholar
  13. 13.
    Ali S, Zakim D: The effects of bilirubin on the thermal properties of phosphatidylcholine bilayers. Biophys J 65: 101–105, 1993PubMedGoogle Scholar
  14. 14.
    Zucker SD, Goessling W, Zeidel ML, Gollan JL: Membrane lipid composition and vesicle size modulate bilirubin intermembrane transfer. Evidence for membrane directed trafficking of bilirubin in the hepatocyte. J Biol Chem 269: 19262–19270, 1994PubMedGoogle Scholar
  15. 15.
    Odell GB: The distribution of bilirubin between albumin and mitochondria. J Pediatr 68: 164–180, 1966Google Scholar
  16. 16.
    Kaufmann NA, Simcha AJ, Blondheim SH: The uptake of bilirubin by blood cells from plasma and its relationship to the criteria for exchange transfusion. Clin Sci 33: 201–208, 1967PubMedGoogle Scholar
  17. 17.
    Karp WB: Biochemical alterations in neonatal hyperbilirubinemia and bilirubin encephalopathy: A review. Pediatrics 64: 361–368, 1979PubMedGoogle Scholar
  18. 18.
    Karp WB, Subramanyam SB, Ho CK, Robertson AF: Drugs affecting bilirubin uptake by human erythrocyte ghosts. Am J Med Sci 289: 236–239, 1985PubMedGoogle Scholar
  19. 19.
    Wennberg RP: The importance of free bilirubin acid salt in bilirubin uptake by erythrocytes and mitochondria. Pediatr Res 23: 443–447, 1988PubMedGoogle Scholar
  20. 20.
    Zucker SD, Storch J, Zeidel ML, Gollan JL: Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles. Biochemistry 31: 3184–3192, 1992CrossRefPubMedGoogle Scholar
  21. 21.
    Brito MA, Silva RM, Matos DC, De Silva AT, Brites DT: Alterations of erythrocyte morphology and lipid composition by hyperbilirubinemia. Clin Chim Acta 249: 149–165, 1996CrossRefPubMedGoogle Scholar
  22. 22.
    Zakim D, Wong PTT: A high-pressure infrared spectroscopic study of the solvation of bilirubin in lipid bilayers. Biochemistry 29: 2003–2007, 1990CrossRefPubMedGoogle Scholar
  23. 23.
    Rashid H, Ali MK, Tayyab S: Differential accessibility of bilirubin to erythrocyte membrane vesicles bearing different structural features. Comp Biochem Physiol 127: 345–350, 2000Google Scholar
  24. 24.
    Lamola AA, Eisinger J, Blumberg WE, Pathel SC, Flores J: Fluorimetric study of the partition of bilirubin among blood components. Basis for rapid micro assays of bilirubin and bilirubin binding capacity in whole blood. Anal Biochem 100: 25–42, 1979CrossRefPubMedGoogle Scholar
  25. 25.
    Kaul R, Bajpai VK, Shipstone AC, Kaul MK, Murti CRK: Bilirubin-induced erythrocyte membrane cytotoxicity. Exp Mol Pathol 34: 290–298, 1981CrossRefPubMedGoogle Scholar
  26. 26.
    Hansen TWR, Odden JP, Bratlid D: Effects of hyperoxia on entry of bilirubin and albumin into rat brain. J Perinatol 7: 217–220, 1987PubMedGoogle Scholar
  27. 27.
    Hansen TWR, Oyasaeter S, Stiris T, Bratlid D: Effects of sulfisoxazole, hypercarbia and hyperosmolality on entry of bilirubin and albumin into brain regions in young rats. Biol Naonate 56: 22–30, 1989Google Scholar
  28. 28.
    Gourley GR: Bilirubin metabolism and kernicterus. Adv Pediatr 44: 173–229, 1997PubMedGoogle Scholar
  29. 29.
    Blauer G, Harmatz D, Snir J: Optical properties of bilirubin-serum albumin complexes in aqueous solution. I. Dependence on pH. Biochim Biophys Acta 278: 68–88, 1972Google Scholar
  30. 30.
    Blauer G, Harmatz D: Optical properties of bilirubin-serum albumin complexes in aqueous solution. II. Effects of electrolytes and of concentration. Biochim Biophys Acta 278: 89–100, 1972PubMedGoogle Scholar
  31. 31.
    Beaven GH, D’Albis A, Gratzer WB: The interaction of bilirubin with human serum albumin. Eur J Biochem 33: 500–510, 1973CrossRefPubMedGoogle Scholar
  32. 32.
    Harmatz D, Blauer G: Optical properties of bilirubin-serum albumin complexes in aqueous solution. A comparison among albumins from different species. Arch Biochem Biophys 170: 375–383, 1975CrossRefPubMedGoogle Scholar
  33. 33.
    Lightner DA, Reisinger M, Landen GL: On the structure of albumin-bound bilirubin. J Biol Chem 261: 6034–6038, 198Google Scholar
  34. 34.
    Lightner DA, Wijekoon WMD, Zhang MH: Understanding bilirubin conformation and binding. J Biol Chem 263: 16669–16676, 1988PubMedGoogle Scholar
  35. 35.
    Brodersen R: Competitive binding of bilirubin and drugs to human serum albumin studied by enzymatic oxidation. J Clin Invest 54: 1353–1364, 1974PubMedGoogle Scholar
  36. 36.
    Brodersen R, Ebbesen F: Bilirubin displacing effect of ampicillin, indomethacin, chlorpromazine, gentamicin and parabens in vitro and in newborn infants. J Pharm Sci 72: 248–253, 1983PubMedGoogle Scholar
  37. 37.
    Brodersen R, Robertson A: Ceftriaxone binding to human serum albumin. Competition with bilirubin. Mol Pharmacol 36: 478–483, 1989Google Scholar
  38. 38.
    Robertson A, Karp W, Brodersen R: Bilirubin displacing effect of drugs used in neonatology. Acta Paediatr Scand 80: 1119–1127, 1991PubMedGoogle Scholar
  39. 39.
    Brodersen R: Binding of bilirubin and other ligands to human serum albumin. In: T. Peters, I. Sjoholm (eds). Albumin Structure, Biosynthesis, Function. Pergamon Press, New York, 1978, Vol. 50, pp. 61–70Google Scholar
  40. 40.
    Sheetz MP, Singer SJ: Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions. Proc Nat Acad Sci, USA 71: 4457–4461, 1974Google Scholar
  41. 41.
    McDonagh AF, Assisi F: The ready isomerization of bilirubin IX-α in aqueous solution. Biochem J 129: 797–800, 1972PubMedGoogle Scholar
  42. 42.
    MsDonagh AF, Assisi F: Commercial bilirubin: A trinity of isomers. FEBS Lett 18: 315–317, 1971CrossRefPubMedGoogle Scholar
  43. 43.
    Tayyab S, Qasim MA: Purification and properties of buffalo serum albumin. Biochem Int 20: 405–415, 1990PubMedGoogle Scholar
  44. 44.
    Chen RF: Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem 242: 173–181, 1967PubMedGoogle Scholar
  45. 45.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurements with the Folin-phenol reagent. J Biol Chem 193: 265–275, 1951PubMedGoogle Scholar
  46. 46.
    Steck TL, Kant JA: Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Methods Enzymol 31: 172–180, 1973Google Scholar
  47. 47.
    Fog J: Determination of bilirubin in serum as alkaline ‘azobilirubin’. Scand J Clin Lab Invest 10: 241–245, 1958PubMedGoogle Scholar
  48. 48.
    Gray CH, Kulczycka A, Nicholson DC: The chemistry of bile pigments. IV. Spectrophotometric titration of the bile pigments. J Chem Soc 442: 2268–2275, 1961Google Scholar
  49. 49.
    Blauer G, Lavie E, Silfen J: Relative affinities of bilirubin for serum albumins from different species. Biochim Biophys Acta 492: 64–69, 1977PubMedGoogle Scholar
  50. 50.
    Peters T Jr: Serum albumin. Adv Protein Chem 37: 161–245, 1985PubMedGoogle Scholar
  51. 51.
    Woolley PV, Hunter MJ: Binding and circular dichroism data on bilirubin-albumin in the presence of oleate and salicylate. Arch Biochem Biophys 140, 197–209, 1970CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Huma Rashid
    • 1
  • Mohammad Mushahid Khan
    • 1
    • 2
  • Saad Tayyab
    • 1
    • 3
  1. 1.Interdisciplinary Biotechnology UnitAligarh Muslim UniversityAligarhIndia
  2. 2.Department of Neurology, School of MedicineThe Medical College of GeorgiaAugustaUSA
  3. 3.Insitut Sains BiologiUniversiti MalayaKuala LumpurMalaysia

Personalised recommendations