Dissolved organic matter in water of Daugava river

Monitoring of Free Radical Processes in Protein-lipid Systems with a NewSquaraine DyeO.Kutsenko *1 , V.Trusova 1 , G.Gorbenko 1 , T.Deligeorgiev 2 , E.Slobozhanina 3 , L.Lukyanenko 3 ,G.Zubritskaya 31 V.N. Karazin Kharkov National University, Kharkov, Ukraine;2 Faculty of Pharmacy and Chemistry, University of Sofia, Sofia, Bulgaria;3 Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus,Minsk, Belaruse-mail: olzk@mail.ruAmyloids are highly ordered protein aggregates whose deposition in organs and tissues is oneof the characteristics of so called protein misfolding diseases. These disorders includeParkinson, Alzheimer, Huntington, prion diseases, etc. It was demonstrated that one of thereasons of amyloid cytotoxicity involves interaction of aggregated proteins with cellmembranes, particularly with red blood cell membrane. The mechanisms of erythrocyte injuryinclude modification of membrane fluidity, alterations in 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 containing lysozyme amyloid fibrils (Lz), hemoglobin (Hb)and liposomes from phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) andcholesterol (Chol). For identification of the presence of active oxygen species novel squarainedye SQ-1 was applied [1]. It has been previously found that Hb-induced formation of freeradicals leads to the drastic quenching of SQ-1 fluorescence, resulting 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 indicating insensibility of this probeto Lz-induced membrane modification. Hb addition to liposomes without Lz was followed bysignificant decrease of SQ-1 emission indicating the formation of free radicals. SQ-1fluorescence quenching was found to depend on model membrane composition. In PC andPC/Chol bilayers the decrease of SQ-1 fluorescence was less pronounced than in CLcontainingmembranes. Interaction of the Hb with negatively charged membranes can befollowed by protein unfolding, heme displacement and dissociation of heme-globin complex.This facilitates heme-lipid interaction and consequent iron-induced formation of free radicalsleading to the increase of free radical formation in CL bilayers as compared to PCmembranes. Cholesterol prevents Hb penetration into the inner membrane regions, thusinhibiting oxidative processes. Interestingly, lysozyme fibrils suppressed SQ-1 fluorescencequenching in all types of lipid bilayers, with the magnitude of this effect being mostpronounced in CL-containing membranes. There are at least two explanations for Lzantioxidant effect. First, protein molecular groups can be the scavengers for free radicals. Onthe other hand, Hb and Lz can compete for the membrane binding sites, thereby reducing Hbpenetration into the membrane interior and formation of free radicals.This work was supported by the grant from Fundamental Research State Fund of Ukraine(project number F41.1/014) and Fundamental Research State Fund of Belarus Republic(project number B11K-152).1. Ioffe, V.M., Gorbenko, G.P., Deligeorgiev, T., Gadjev, N., Vasilev, A. Fluorescence study ofprotein-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 Monitoring Oxidative Processes in Lipid Membranes. J. Fluoresc., 2009, vol. 19, p. 1017–1023.- 26 -