Dissolved organic matter in water of Daugava river

Aminobenzanthrone Dyes as Prospective Fluorophores for Detection andCharacterization of Amyloid FibrilsK.Vus* 1 , V.Trusova 1 , G.Gorbenko 1 , E.Kirilova 2 , G.Kirilov 2 , I.Kalnina 21 Department of Biological and Medical Physics, V. N. Karazin Kharkiv National University,Kharkiv, Ukraine;2 Department of Chemistry and Geography, Daugavpils University, Daugavpils, Latviae-mail: katenka.vus@mail.ruIn view of dramatic role of pathological protein aggregates, amyloid fibrils, in thedevelopment of a number of severe diseases, detection and characterization of such entitiesrepresent the focus of current research efforts. Fluorescence spectroscopy has beenextensively employed in amyloid studies due to its inherent sensitivity and versatility, goodspatial and temporal resolution. The aim of this work was to test a series of novel fluorescentaminobenzanthrone dyes (BD) for their ability to identify and characterize fibrillar aggregatesof lysozyme prepared by protein denaturation in concentrated ethanol solution (FI) [1] or atstrongly acidic pH (FII) [2]. Analysis of the results of fluorimetric titration in terms of theLangmuir adsorption model yielded the parameters of BD binding to native and fibrillarprotein (association constant, binding stoichiometry and molar fluorescence). Furthermore,several additional quantities reflecting the preference of the probe to fibrillar proteinaggregates have been evaluated [3]. Based on the comprehensive analysis of the recoveredparameters, AM2 and A8 were selected as the most prospective tracers for FI and FII,respectively. Besides, A6 and A8 were also suitable for highly sensitive detection of both FIand FII types of amyloid fibrils, because of their high quantum yields in amyloid-bound state.Interestingly, comparison of quantum yields and binding parameters of the dyes in FI- andFII-bound states showed the differences in fibrillar morphologies. It was supposed that in FIIfibers protofilaments are packed more tightly than in FI, that complicates BD penetration intothe FII fibrils and is presumably responsible for the decrease of BD-protein binding extent.Finally, strong sensitivity of the novel probes to environmental viscosity was revealed byanalyzing their properties in ethanol (E) and in ethanol/glycerol mixture with glycerol contentof 67% (v/v) (EG). Substantially higher fluorescence anisotropy of BD in EG compared to Eis indicative of strong viscosity sensitivity of the novel dyes. These findings suggest thatfluorescence anisotropy of BD can be employed as an alternative parameter for studying thekinetics of amyloid fibril formation [4]. The present study demonstrated high potential ofaminobenzanthrone dyes in identification of fibrillar protein aggregates and uncovering theirstructural peculiarities.This work was supported by the grants from European Social Fund (project number2009/0205/1DP/1.1.1.2.0/09/APIA/VIAA/152) and Fundamental Research State Fund ofUkraine (project number F.41.4/014).References:1. Holley, M., Eginton, C., Schaefer, D., Brown, L.R. Characterization of Amyloidogenesis of HenEgg Lysozyme in Concentrated Ethanol Solution. Biochem Biophys Res Commun, 2008, vol. 373,p. 164-168.2. Morozova-Roche, L.A., Zurdo, J., Spencer, A., Noppe, W., Receveur, V., Archer, D.B., Joniau,M., Dobson, C.M. Amyloid Fibril Formation and Seeding by Wild-Type Human Lysozyme andIts Disease-Related Mutational Variants. J Struct Biol, 2000, vol. 130, p. 339-351.3. Vus, K., Trusova, V., Gorbenko, G., Kirilova, E., Kirilov, G., Kalnina, I., Kinnunen, P. NovelAminobenzanthrone Dyes for Amyloid Fibril Detection. Chem Phys Lett, 2010, vol. 532, p. 110-115.4. Sabate, R., Saupe, S.J. Thioflavin T Fluorescence Anisotropy: an Alternative Technique for theStudy of Amyloid Aggregation. Biochem Biophys Res Commun, 2007, vol. 360, p. 135-138.- 33 -