V. Trusova, G. Gorbenko, U. Tarabara, K. Vus, O. Ryzhova


The protolytic and partition equilibria of the indicator dyes in the model lipid and protein-lipid systems have been analyzed. A methodological approach has been developed allowing the partition coefficients of the protonated and deprotonated dye forms to be derived from the spectrophotometric measurements. The partitioning of the indicator dye bromothymol blue into the model bilayer membranes composed of phosphatidylcholine and cardiolipin (9:1, mol:mol) has been examined. The partition coefficient of the protonated dye species into a lipid phase has been found to be 5 orders of magnitude higher than that of the deprotonated dye form. This effect has been interpreted in terms of the differences in the charge distribution over the protonated and deprotonated dye ions, preventing the hydrophobic dye-lipid interactions in the latter case. The reduction of the bromothymol blue partitioning into lipid bilayer in the presence of hemoglobin has been attributed to the protein-induced changes in the structure and physicochemical characteristics of the interfacial membrane region. In the practical aspect, the obtained findings may prove of significance in the design of hemosome-based blood substitutes and elucidating the role of hemoglobin in the molecular etiology of the amyloid disorders, particularly, Alzheimer's disease.


indicator dye; partition coefficient; liposomes; hemoglobin; protein-lipid interaction

Full Text:



White S., Ladokhin A., Jayasinghe S., Hristova K. How membranes shape protein structure // J. Biol. Chem. – 2001. – Vol. 276. – P. 32395-32398.

Mouritsen O. Self-assembly and organization of lipid - protein membranes // Current Opin. Colloid Interface Sci. – 1998. – Vol. 3. – P. 78-87.

Denisov G., Wanaski S., Luan P., Glaser M., McLaughlin S. Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-biphosphate: an electrostatic model and experimental results // Biophys. J. – 1998. – Vol. 74. – P. 731-744.

Ben-Tal N., Honig B., Miller C., McLaughlin S. Electrostatic binding of proteins to membranes. Theoretical predictions and experimental results with charybdotoxin and phospholipid vesicles // Biophys. J. – 1997. – Vol. 73. – P. 1717-1727.

Sankaram M., Marsh D. Protein-lipid interactions with peripheral membrane proteins // Protein-Lipid Interactions / Ed. by A.Watts. – Elsevier, 1993. – P. 127-162.

Kleinschmidt J., Mahaney J., Thomas D., Marsh D. Interaction of bee venom melittin with zwitterionic and negatively charged phospholipid bilayers: a spin - label electron spin resonance study // Biophys. J. – 1997. – Vol. 72. – P. 767-778.

Dumas F., Lebrun M., Peyron P., Lopez A., Tocanne J. The transmembrane protein bacterioopsin affects the polarity of the hydrophobic core of the host lipid bilayer // Biochim. Biophys. Acta. – 1999. – Vol. 1421. – P. 295-305.

Roux M., Newmann Y., Hodges R. Conformational changes of phospholipid headgroups induced by a cationic integral membrane peptide as seen by deuterium magnetic resonance // Biochem. – 1989. – Vol. 28. – P. 2313-2321.

Dempsey C., Bitbol M., Watts A. Interaction of melittin with mixed phospholipid membranes composed of dimyristoylphosphatidylserine studied by deuterium NMR // Biochem. – 1989. – Vol. 28. – P. 6590-6595.

Babin Y., D’Amour J., Pigeon M., Pezolet M. A study of the structure of polymyxin B – dipalmitoylphosphatidylglycerol complexes by vibrational spectroscopy // Biochim. Biophys. Acta. – 1987. – Vol. 903. – P. 78-88.

Schwarz G., Beschiachvili G. Thermodynamic and kinetic studies on the association of melittin with a phospholipid bilayer // Biochim. Biophys. Acta. – 1989. – Vol. 979. – P. 82-90.

Moller J., Kragh-Hansen U. Indicator dyes as probes of electrostatic potential changes on macromolecular surfaces // Biochem. – 1975. – Vol. 14. – P. 2317-2323.

Mashimo T., Uede I. Hydrophilic region of lecithin membranes studied by bromothymol blue and effect of inhalation anesthetic, enflurane // Proc. Natl. Acad. Sci. USA. – 1979. – Vol. 76. – P. 5114-5118.

Gorbenko G., Mchedlov-Petrossyan N., Chernaya T. Ionic equilibria in microheterogeneous systems. Protolytic behaviour of indicator dyes in mixed phosphatidylcholine – diphosphatidylglycerol liposomes // J. Chem. Soc. Faraday

Trans. – 1998. – Vol. 94. – P. 2117-2125.

Cevc G. Membrane electrostatics // Biochim. Biophys. Acta. – 1990. – Vol. 1031. – P. 311-382.

Gorbenko G. Bromothymol blue as a probe for structural changes of model membranes induced by hemoglobin // Biochim. Biophys. Acta. – 1998. – Vol. 1370. – P. 107-118.

Batzri S., Korn E. Single bilayer liposomes prepared without sonication // Biochim. Biophys. Acta. – 1973. – Vol. 298. – P. 1015-1019.

Bartlett G. Phosphorus assay in column chromatography // J. Biol. Chem. – 1959. – Vol. 234. – P. 466-468.

Antonini E., Wyman J., Moretti R., Rossi-Fanelli A. The interaction of bromothymol blue with hemoglobin and its effect on the oxygen equilibrium // Biochim. Biophys. Acta. – 1963. – Vol. 71. – P. 124-138.

Benesch R., Benesch E., Yung S. Equations for the spectrophotometric analysis of hemoglobin mixtures // Anal. Biochem – 1973. – Vol. 55. – P. 245-248.

Tocanne J., Teissie J. Ionization of phospholipids and phospholipid - supported interfacial lateral diffusion of protons in membrane model systems // Biochim. Biophys. Acta. – 1990. – Vol. 1031. – P. 111-142.

Ivkov V.G., Berestovsky G.N. Dynamic Structure of Lipid Bilayer. - Moscow: Nauka, 1981.

Cantor C.R., Shimmel P.R. Biophysical Chemistry, Part 2. - San Francisco: W.H. Freeman and Company, 1980.

Johnson M. Parameter correlations while curve fitting // Meth. Enzymol. – 2000. – Vol. 321. – P. 424–446.

Zekany L., Nagypal I. Computational methods for the determination of formation constants. - New York: Plenum Press, 1985.

Pitcher III W., Keller S., Huestis W. Interaction of nominally soluble proteins with phospholipid monolayers at the air-water interface // Biochim. Biophys. Acta. – 2002. – Vol. 1564. – P. 107-113.

Szebeni J., Hauser H., Eskelson C., Watson R., Winterhalter K. Interaction of hemoglobin derivatives with liposomes. Membrane cholesterol protects against the changes of hemoglobin // Biochem. – 1988. – Vol. 27. – P. 6425-6434.

Shaklai N., Yguerabide J., Ranney H. Classification and localization of hemoglobin binding sites on the red blood cell

membrane // Biochem. – 1977. – Vol. 16. – P. 5593-5597.

Szebeni J., Di Lorio E., Hauser H., Winterhalter K. Encapsulation of hemoglobin in phospholipid liposomes: characterization and stability // Biochem. – 1985. – Vol. 24. – P. 2827-2832.

Marva E., Hubbel R. Denaturing interaction between sickle hemoglobin and phosphatidylserine liposomes // Blood. – 1994. – Vol. 83. – P. 242-249.

Shviro Y., Zilber I., Shaklai N. The interaction of hemoglobin with phosphatidylserine vesicles // Biochim. Biophys. Acta. – 1982. – Vol. 687. – P. 63-70.

Bossi L., Alema S., Calissano P., Marra E. Interaction of different forms of hemoglobin with artificial lipid membranes // Biochim. Biophys. Acta. – 1975. – Vol. 375. – P. 477-482.

Chupin V., Ushakova I., Bondarenko S., Vasilenko I., Serebrennikova G., Evstigneeva R., Rosenberg G., Koltsova G. 31P-NMR study of methemoglobin interaction with model membranes // Bioorg. Chem. – 1982. – Vol. 9. – P. 1275-1280.

Gutteridge J. Age pigments and free radicals: fluorescent lipid complexes formed by iron and copper-containing proteins // Biochim. Biophys. Acta. – 1985. – Vol. 834. – P. 144-148.

Gross E., Bedlack R., Loew L. Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential // Biophys. J. – 1994. – Vol. 67. – P. 208-216.

Flewelling R., Hubbel W. The membrane dipole potential in a total membrane potential model. Application to hydrophobic ion interaction with membranes // Biophys. J. – 1986. – Vol. 49. – P. 541-552.

Colonna R., Del’Antone P., Azzone G. Binding changes and apparent pKa shifts of bromothymol blue as tools for mitochondrial reactions // Arch. Biochem. Biophys. – 1972. – Vol. 151. – P. 295-303.

Beschiaschvili G., Seelig J. Melittin binding to mixed phosphatidylglycerol - phosphatidylcholine membrane // Biochem. – 1990. – Vol. 29. – P. 52-58.

Wu C.W., Liao P.C., Yu L., Wang S.T., Chen S.T., Wu C.M., Kuo Y.M. Hemoglobin promotes AB oligomer formation and localizes in neurons and amyloid deposits // Neurobiology of Disease. – 2004. – Vol. 17. – P. 367-377.

Bishop G.M., Robinson S.R., Liu Q., Perry G., Atwood C.S., Smith M.A. Iron: a pathological mediator of Alzheimer disease // Dev. Neurosci. – 2002. – Vol. 24. – P. 184–187.

Kutsenko O.K., Trusova V.M., Gorbenko G.P., Lipovaya A.S., Slobozhanina E.I., Lukyanenko L.M., Deligeorgiev T., Vasilev A. Fluorescence Study of the Membrane Effects of Aggregated Lysozyme // J. Fluoresc. – 2013. – Vol. 23. – P. 1229–1237.


  • There are currently no refbacks.

Copyright (c) 2017 V. Trusova, G. Gorbenko, U. Tarabara, K. Vus, O. Ryzhova