Monitor<strong>in</strong>g <strong>of</strong> Free Radical Processes <strong>in</strong> Prote<strong>in</strong>-lipid Systems with a NewSquara<strong>in</strong>e DyeO.Kutsenko *1 , V.Trusova 1 , G.Gorbenko 1 , T.Deligeorgiev 2 , E.Slobozhan<strong>in</strong>a 3 , L.Lukyanenko 3 ,G.Zubritskaya 31 V.N. Karaz<strong>in</strong> Kharkov National University, Kharkov, Ukra<strong>in</strong>e;2 Faculty <strong>of</strong> Pharmacy and Chemistry, University <strong>of</strong> S<strong>of</strong>ia, S<strong>of</strong>ia, Bulgaria;3 Institute <strong>of</strong> Biophysics and Cell Eng<strong>in</strong>eer<strong>in</strong>g, National Academy <strong>of</strong> Sciences <strong>of</strong> Belarus,M<strong>in</strong>sk, Belaruse-mail: olzk@mail.ruAmyloids are highly ordered prote<strong>in</strong> aggregates whose deposition <strong>in</strong> organs and tissues is one<strong>of</strong> the characteristics <strong>of</strong> so called prote<strong>in</strong> misfold<strong>in</strong>g diseases. These disorders <strong>in</strong>cludePark<strong>in</strong>son, Alzheimer, Hunt<strong>in</strong>gton, prion diseases, etc. It was demonstrated that one <strong>of</strong> thereasons <strong>of</strong> amyloid cytotoxicity <strong>in</strong>volves <strong>in</strong>teraction <strong>of</strong> aggregated prote<strong>in</strong>s with cellmembranes, particularly with red blood cell membrane. The mechanisms <strong>of</strong> erythrocyte <strong>in</strong>jury<strong>in</strong>clude modification <strong>of</strong> membrane fluidity, alterations <strong>in</strong> enzyme activity and oxidation state,apoptosis and oxidative stress.The present study was undertaken to explore amyloid effect on membrane lipid oxidation. Tothis end, we used model systems conta<strong>in</strong><strong>in</strong>g lysozyme amyloid fibrils (Lz), hemoglob<strong>in</strong> (Hb)and liposomes from phosphatidylchol<strong>in</strong>e (PC) and its mixtures with cardiolip<strong>in</strong> (CL) andcholesterol (Chol). For identification <strong>of</strong> the presence <strong>of</strong> active oxygen species novel squara<strong>in</strong>edye SQ-1 was applied [1]. It has been previously found that Hb-<strong>in</strong>duced formation <strong>of</strong> freeradicals leads to the drastic quench<strong>in</strong>g <strong>of</strong> SQ-1 fluorescence, result<strong>in</strong>g presumably from thereaction between reactive oxygen species and SQ-1 squaric moiety [2]. Lz association withlipid bilayers was not followed by SQ-1 spectral changes <strong>in</strong>dicat<strong>in</strong>g <strong>in</strong>sensibility <strong>of</strong> this probeto Lz-<strong>in</strong>duced membrane modification. Hb addition to liposomes without Lz was followed bysignificant decrease <strong>of</strong> SQ-1 emission <strong>in</strong>dicat<strong>in</strong>g the formation <strong>of</strong> free radicals. SQ-1fluorescence quench<strong>in</strong>g was found to depend on model membrane composition. In PC andPC/Chol bilayers the decrease <strong>of</strong> SQ-1 fluorescence was less pronounced than <strong>in</strong> CLconta<strong>in</strong><strong>in</strong>gmembranes. Interaction <strong>of</strong> the Hb with negatively charged membranes can befollowed by prote<strong>in</strong> unfold<strong>in</strong>g, heme displacement and dissociation <strong>of</strong> heme-glob<strong>in</strong> complex.This facilitates heme-lipid <strong>in</strong>teraction and consequent iron-<strong>in</strong>duced formation <strong>of</strong> free radicalslead<strong>in</strong>g to the <strong>in</strong>crease <strong>of</strong> free radical formation <strong>in</strong> CL bilayers as compared to PCmembranes. Cholesterol prevents Hb penetration <strong>in</strong>to the <strong>in</strong>ner membrane regions, thus<strong>in</strong>hibit<strong>in</strong>g oxidative processes. Interest<strong>in</strong>gly, lysozyme fibrils suppressed SQ-1 fluorescencequench<strong>in</strong>g <strong>in</strong> all types <strong>of</strong> lipid bilayers, with the magnitude <strong>of</strong> this effect be<strong>in</strong>g mostpronounced <strong>in</strong> CL-conta<strong>in</strong><strong>in</strong>g membranes. There are at least two explanations for Lzantioxidant effect. First, prote<strong>in</strong> molecular groups can be the scavengers for free radicals. Onthe other hand, Hb and Lz can compete for the membrane b<strong>in</strong>d<strong>in</strong>g sites, thereby reduc<strong>in</strong>g Hbpenetration <strong>in</strong>to the membrane <strong>in</strong>terior and formation <strong>of</strong> free radicals.This work was supported by the grant from Fundamental Research State Fund <strong>of</strong> Ukra<strong>in</strong>e(project number F41.1/014) and Fundamental Research State Fund <strong>of</strong> Belarus Republic(project number B11K-152).1. I<strong>of</strong>fe, V.M., Gorbenko, G.P., Deligeorgiev, T., Gadjev, N., Vasilev, A. Fluorescence study <strong>of</strong>prote<strong>in</strong>-lipid complexes with a new symmetric squarylium probe. Biophys. Chem., 2007, vol. 128, p.75–86.2. Trusova, V.M., Gorbenko, G.P., Deligeorgiev, T., Gadjev, N., Vasilev, A. A Novel Squarylium Dyefor Monitor<strong>in</strong>g Oxidative Processes <strong>in</strong> Lipid Membranes. J. Fluoresc., 2009, vol. 19, p. 1017–1023.- 26 -
Study<strong>in</strong>g Z<strong>in</strong>c Biology with Fluorescent ProbesE.I.Slobozhan<strong>in</strong>a *1 ,Y.M.Harmaza 1 , N.M. Kozlova 1 , A.V.Tamashevski 11 Institute <strong>of</strong> biophysics and cell eng<strong>in</strong>eer<strong>in</strong>g <strong>of</strong> NAS B, M<strong>in</strong>sk, Belaruse-mail: slobozhan<strong>in</strong>a@ibp.org.byBackground: Zn 2+ is essential trace element that is required for a wide range <strong>of</strong> biologicalprocesses, <strong>in</strong>clud<strong>in</strong>g cell proliferation and differentiation. The physiological concentration<strong>of</strong> Zn 2+ <strong>in</strong> serum is 2–15 μM, when free Zn 2+ level <strong>in</strong> most cells is extremely low (
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