Interaction of bilirubin with native and protein-depleted human erythrocyte membranes

  • Huma Rashid
  • Saad Tayyab
Part of the Molecular and Cellular Biochemistry: An International Journal for Chemical Biology in Health and Disease book series (DMCB, volume 41)


In order to study the role of membrane proteins in bilirubin (BR) binding phenomenon, selective removal of membrane proteins was carried out using various reagents, namely, ethylenediamine tetraacetic acid (EDTA), sodium hydroxide (NaOH), 3,5-diiodosalicylic acid, lithium salt (LIS), dimethylmaleic anhydride (DMMA), sodium iodide (NaI), o-phenanthroline-cupric sulfate (CuP) and phenanthroline-cupric sulfate containing 2-mercaptoethanol (CuP-mercaptoethanol). Effects of these treatments on the conformation and BR binding properties of the membrane were studied using circular dichroism (CD) spectroscopy as well as estimation of membrane-bound BR by diazotised-color reaction. Though a significant amount of protein (ranging from 23-69%) was lost from the membranes upon these treatments, only a small decrease (3-13%) was observed in BR binding, being maximum with NaOH-treated membranes. However, DMMA and NaI treatments produced a little increase in BR binding. Conformation of the membrane was retained to a significant extent as indicated by far-UV CD spectra upon these treatments except in DMMA and Nat treatments which resulted in the perturbation in CD spectra. Taken together, these results suggest that membrane proteins play little role in BR binding, rather act as barriers in BR binding phenomenon. (Mol Cell Biochem 246: 171–177, 2003)

Key words

human erythrocyte membrane membrane proteins cross-linking bilirubin binding CD spectra 


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  1. 1.
    Sato H, Kashiwamata S: Interaction of bilirubin with human erythrocyte membranes. Biochem J 210: 489–496, 1983PubMedGoogle Scholar
  2. 2.
    Sato H, Aono S, Semba R, Kashiwamata S: Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidaseand phospholipase-treated membranes. Biochem J 248: 21–26, 1987PubMedGoogle Scholar
  3. 3.
    Gulian J-M, Gonard V, Dalmasso C, Palix C: Bilirubin displacement by ceftriaxone in neonates: Evaluation by determination of ‘free’ bilirubin and erythrocyte-bound bilirubin. J Antimicrobial Chemother 19: 823–829, 1987CrossRefGoogle Scholar
  4. 4.
    Hayer M, Piva MT, Sieso V, De Bornier BM: Experimental studies on unconjugated bilirubin binding by human erythrocytes. Clin Chim Acta 186: 345–350, 1989CrossRefGoogle Scholar
  5. 5.
    Nagaoka S, Cowger ML: Interaction of bilirubin with lipids studied by fluorescence quenching method. J Biol Chem 253: 2005–2011, 1978PubMedGoogle Scholar
  6. 6.
    Cestaro B, Cervato G, Ferrari S, Di Silvestro G, Monti D, Manitto P: Interaction of bilirubin with small unilamellar vesicles of dipalmitoylphosphatidylcholine. Ital J Biochem 32: 318–329, 1983PubMedGoogle Scholar
  7. 7.
    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
  8. 8.
    Leonard M, Noy N, Zakim D: The interaction of bilirubin with model and biological membranes. J Biol Chem 264: 5648–5652, 1989PubMedGoogle Scholar
  9. 9.
    Sheetz MP, Wang WP, Kreutzer DL: Polyphosphoinositides as regulators of membrane skeletal stability. Kroc Found Ser 16: 87–94, 1984PubMedGoogle Scholar
  10. 10.
    Haest CWM: Interaction between membrane skeleton proteins and the intrinsic domain of the erythrocyte membrane. Biochim Biophys Acta 694: 331–352, 1982PubMedCrossRefGoogle Scholar
  11. 11.
    Rodgers W, Glaser M: Distribution of proteins and lipids in the erythrocyte membrane. Biochemistry 32: 12591–12598, 1993PubMedCrossRefGoogle Scholar
  12. 12.
    Kumar A, Gupta CM: Red cell membrane abnormalities in chronic myeloid leukaemia. Nature 303: 632–633, 1983PubMedCrossRefGoogle Scholar
  13. 13.
    Steck TL: The organization of proteins in the human red blood cell membrane. A review. J Cell Biol 62: 1–19, 1974PubMedCrossRefGoogle Scholar
  14. 14.
    Palfrey FIC, Waseem A: Protein kinase C in the human erythro-cyte. Translocation to the plasma membrane proteins. J Biol Chem 260: 16021–16029, 1985PubMedGoogle Scholar
  15. 15.
    Rashid H, Owais M, Tayyab S: Bilirubin binding to normal and modified human erythrocyte membranes. Effect of phospholipases, neuraminidase, trypsin and CaCl2Mol Cell Biochem 228: 15–23, 2001CrossRefGoogle Scholar
  16. 16.
    Kahlenberg A: Partial purification of a membrane protein from human erythrocytes involved in glucose transport. J Biol Chem 251: 1582–1590, 1972Google Scholar
  17. 17.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970PubMedCrossRefGoogle Scholar
  18. 18.
    Lowry OH, Rosebrough NJ, Farr AL Randall RJ: Protein measurements with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951PubMedGoogle Scholar
  19. 19.
    Fog J: Determination of bilirubin in serum as alkaline `azobilirubin’. Scan J Clin Lab Invest 10: 241–245, 1958Google Scholar
  20. 20.
    Tayyab S, Ali MK: A comparative study on the extraction of membrane-bound bilirubin from erythrocyte membranes using various methods. J Biochem Biophys Meth 39: 39–45, 1999PubMedCrossRefGoogle Scholar
  21. 21.
    Van Hoek AN, Wiener M, Bicknese S, Miercke L, Biwersi J, Verkman AS: Secondary structure analysis of purified functional CH1P28 water channels by CD and FTIR spectroscopy. Biochemistry 32: 11847–11856. 1993PubMedCrossRefGoogle Scholar
  22. 22.
    Fairbanks G, Steck TL, Wallach DFH: Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10: 2606–2617, 1971PubMedCrossRefGoogle Scholar
  23. 23.
    Steck TL, Kant JA: Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Meth Enzymol 31: 172–180, 1973CrossRefGoogle Scholar
  24. 24.
    Jones MN, Nickson JK: Monosaccharide transport proteins of the human erythrocyte membrane. Biochim Biophys Acta 650: 1–20, 1981PubMedCrossRefGoogle Scholar
  25. 25.
    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
  26. 26.
    Marchesi VT, Andrews EP: Glycoproteins. Isolation from cell membranes with lithium diiodosalicylate. Science 174: 1247–1248, 1971PubMedCrossRefGoogle Scholar
  27. 27.
    Makino S, Nakashima HJ: Behaviour of fragmented Band 3 from chymotrypsin-treated bovine erythrocyte membrane in non-ionic detergent solution. J Biochem 92: 1069–1077, 1982PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2003

Authors and Affiliations

  • Huma Rashid
    • 1
  • Saad Tayyab
    • 1
  1. 1.Interdisciplinary Biotechnology UnitAligarh Muslim UniversityAligarhIndia

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