Abstract
Binding of bilirubin to human erythrocyte membranes was studied after various enzymatic treatments as well as calcium loading. Whereas phospholipase D treatment of erythrocyte membranes resulted in 23% increase in bilirubin binding, phospholipase C-treated membranes showed remarkable enhancement in bilirubin binding. Polar head groups in general and negatively charged phosphate moieties, in particular, of phospholipids of the membrane appear to inhibit a large amount of bilirubin from binding to the membranes. Neuraminidase treatment of the membranes also led to a slight increase in bilirubin binding as compared to untreated membranes. Membrane proteins and carbohydrates seem to play significant regulatory role in bilirubin binding. However, no direct correlation was found between the increase in bilirubin binding and the amount of carbohydrate released upon tryptic digestion of membranes. Increase in bilirubin binding to trypsin-treated membranes can be ascribed to the increase in free bilirubin concentration in the incubation mixture as a result of tryptic hydrolysis of albumin by the membrane-bound tryptic activity. Calcium-loaded erythrocyte membranes also showed remarkable increase in bilirubin binding as a result of negative charge shielding and calcium-induced hydrophobic aggregation. Taken together, these results suggest the inhibitory role of polar head groups of phospholipids (phosphate in particular), carbohydrate and sialic acid in the binding of bilirubin to erythrocyte membranes.
Similar content being viewed by others
References
Wennberg RP, Ahlfors CE, Rasmussen LF: The pathochemistry of kernicterus. Early Hum Dev 3: 353–372, 1979
Eriksen EF, Danielsen H, Brodersen R: Bilirubin-liposome interaction. Binding of bilirubin dianion, protonization and aggregation of bilirubin acid. J Biol Chem 256: 4269–4274, 1981
Cashore WJ, Oh W: Kernicterus and bilirubin encephalopathy. Semin Liver Dis 8: 163–167, 1988
Odell GB: Influence of pH on the distribution of bilirubin between albumin and mitochondria. Proc Soc Exp Biol Med 120: 352–354, 1965
Odell GB: The distribution of bilirubin between albumin and mitochondria. J Pediatr 68: 164–180, 1966
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, 1967
Bratlid D: Bilirubin binding by human erythrocytes. Scand J Clin Lab Invest 29: 91–97, 1972
Odell GB: Toxicity of bilirubin and assessment of its risk during neonatal life. In: T.K. Oliver (ed). Neonatal Hyperbilirubinemia. Grune and Straton, New York, 1980, pp 83–113
Karp WB, Subramanyam SB, Ho CK, Robertson AF: Drugs affecting bilirubin uptake by human erythrocyte ghosts. Am J Med Sci 289: 236–239, 1985
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, 1992
Zakim D, Wong PTT: A high-pressure, infrared spectroscopic study of the solvation of bilirubin in lipid bilayers. Biochemistry 29: 2003–2007, 1990
Brito MA, Silva RM, Matos DC, da Silva AT, Brites DT: Alterations of erythrocyte morphology and lipid composition by hyperbilirubinemia. Clin Chim Acta 249: 149–165, 1996
Sato H, Kashiwamata S: Interaction of bilirubin with human erythrocyte membranes. Biochem J 210: 489–496, 1983
Wennberg RP: The importance of free bilirubin acid salt in bilirubin uptake by erythrocytes and mitochondria. Pediatr Res 23: 443–447, 1988
Hayer M, Piva MT, Sieso V, de Bornier BM: Experimental studies on unconjugated bilirubin binding by human erythrocytes. Clin Chim Acta 186: 345–350, 1989
Brites D, Silva R, Brito A: Effect of bilirubin on erythrocyte shape and haemolysis under hypotonic, aggregating or non-aggregating conditions and correlation with cell age. Scand J Clin Lab Invest 57: 337–350, 1997
Corchs JL, Corchs MJ, Serrani RE: Unconjugated bilirubin effect on 3Houabain binding to human fetal red cells. Rev Esp Fisiol 50: 5–9, 1994
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, 1987
Rashid H, Ali MK, Tayyab S: Differential accessibility of bilirubin to erythrocyte membrane vesicles bearing different structural features. Comp Biochem Physiol 127: 345–350, 2000
Nagaoka S, Cowger ML: Interaction of bilirubin with lipids studied by fluorescence quenching method. J Biol Chem 253: 2005–2011, 1978
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, 1983
Vazquez J, Garcia-Calvo M, Valdivieso F, Mayor F, Mayor F Jr: Interaction of bilirubin with the synaptosomal plasma membrane. J Biol Chem 263: 1255–1265, 1988
Ali MK, Tayyab S: Effect of phospholipase C, trypsin and neuraminidase on binding of bilirubin to mammalian erythrocyte membranes. Comp Biochem Physiol 129: 355–362, 2001
Burkholder DE, Brecher AS: Interaction between proteases and bovine erythrocyte membranes. Biochim Biophys Acta 282: 135–145, 1972
Leonard M, Noy N, Zakim D: The interactions of bilirubin with model and biological membranes. J Biol Chem 264: 5648–5652, 1989
Tayyab S, Qasim MA: Purification and properties of buffalo serum albumin. Biochem Int 20: 405–415, 1990
Palfrey HC, Waseem A: Protein kinase C in the human erythrocyte. Translocation to the plasma membrane and phosphorylation of bands 4.1 and 4.9 and other membrane proteins. J Biol Chem 260: 16021–16029, 1985
Fairbanks G, Steck TL, Wallach DFH: Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10: 2606–2617, 1971
Chen PS Jr, Toribara TY, Warner H: Microdetermination of phosphorus. Anal Chem 28: 1756–1758, 1956
Kates M, Sastry PS: Phospholipase D. Meth Enzymol 14: 197–203, 1969
Warren L: The thiobarbituric acid assay of sialic acids. J Biol Chem 234: 1971–1975, 1959
Steck TL, Fairbanks G, Wallach DFH: Disposition of the major proteins in the isolated erythrocyte membrane. Proteolytic dissection. Biochemistry 10: 2617–2624, 1971
Svennerholm L: The quantitative estimation of cerebrosides in nervous tissue. J Neurochem 1: 42–45, 1956
De Gier J, Van Deenen LLM: Some lipid characteristics of red cell membranes of various animal species. Biochim Biophys Acta 49: 286–296, 1961
Dittmer JC, Wells MA: Quantitative and qualitative analysis of lipids and lipid components. Meth Enzymol 14: 482–530, 1969
Rouser G, Fleischer S, Yamamoto A: Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5: 494–496, 1969
Bartlett GR: Phosphorus assay in column chromatography. J Biol Chem 234: 466–468, 1959
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurements with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951
Fog J: Determination of bilirubin in serum as alkaline ‘azobilirubin'. Scand J Clin Lab Invest 10: 241–245, 1958
Tayyab S, Ali MK: A comparative study on the extraction of membranebound bilirubin from erythrocyte membranes using various methods. J Biochem Biophys Meth 39: 39–45, 1999
Lenard J, Singer SJ: Structure of membranes: Reaction of red blood cell membranes with phospholipase C. Science 159: 738–739, 1968
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, 1994
Schmidt M, Frings M, Mono ML, Guo Y, Weernink PA, Evellin S, Han L, Jakobs KH: G protein-coupled receptor-induced sensitization of phospholipase C stimulation by receptor tyrosine kinases. J Biol Chem 275: 32603–32610, 2000
Shen Y, Xu L, Foster DA: Role of phospholipase D in receptor-mediated endocytosis. Mol Cell Biol 21: 595–602, 2001
Plo I, Lautier D, Levade T, Sekouri H, Jaffrezou JP, Laurent G, Bettaieb A: Phosphatidylcholine-specific phospholipase C and phospholipase D are respectively implicated in mitogen-activated protein kinase and nuclear factor kappa B activation in tumour necrosis factor alpha-treated immature acute myeloid leukaemia cells. Biochem J 351: 459–467, 2000
Choy YM, Wong SL, Lee CY: Changes in surface carbohydrates of erythrocytes during in vivo aging. Biochem Biophys Res Commun 91: 410–415, 1979
Ito T, Ohnishi S, Ishinaga M, Kito M: Synthesis of a new phosphatidylserine spin-label and calcium-induced lateral phase separation in phosphatidylserine-phosphatidylcholine membranes. Biochemistry 14: 3064–3069, 1975
Kaznacheyeva E, Zubov A, Gusev K, Bezprozvanny I, Mozhayeva GN: Activation of calcium entry in human carcinoma A431 cells by store depletion and phospholipase C-dependent mechanisms converge on ICRAC-like calcium channels. Proc Natl Acad Sci, USA 98: 148–153, 2001
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Rashid, H., Owais, M. & Tayyab, S. Bilirubin binding to normal and modified human erythrocyte membranes: Effect of phospholipases, neuraminidase, trypsin and CaCl2. Mol Cell Biochem 228, 15–23 (2001). https://doi.org/10.1023/A:1013300106220
Issue Date:
DOI: https://doi.org/10.1023/A:1013300106220