Dissolved organic matter in water of Daugava river

ABinding of the Novel 4-tricyanovinylarylamine Dye to Fibrillar LysozymeK.Vus* 1 , V.Trusova 1 , G.Gorbenko 1 , T.Deligeorgiev 2 , S.Kaloyanova 2 , N.Lesev 21 Faculty of Physics and Technology, V. N. Karazin Kharkiv National University, Kharkiv,Ukraine;2 Faculty of Pharmacy and Chemistry, University of Sofia “St. Kliment Ohridsky”, Sofia,Bulgariae-mail: katenka.vus@mail.ruAccumulation of fibrillar protein aggregates (amyloid fibrils) in different tissues is currentlyassociated with a variety of so called conformational diseases. The most widespreadtechniques employed in identification of amyloid fibrils are fluorescence and absorptionspectroscopy. Further progress in this research area is strongly coupled with the design andtesting of the novel chromo- and fluorophores. The present study was aimed at evaluation ofthe novel 4-tricyanovinylarylamine dye (referred here as 3q) as possible marker for fibrillarprotein aggregates. Amyloid fibrils of lysozyme were prepared at strongly acidic pH [1] .Tricyanovinylarylamine probes are characterized by high extinction coefficients and theability to form intramolecular charge transfer state upon excitation [2] . Fluorescent propertiesof these dyes are sensitive to their molecular structure and local environment. It appeared that3q has very low quantum yields in buffer solution and there was no fluorescence increase inthe presence of amyloid fibrils. For this reason, we tested this dye as possible analog of thetraditional amyloid marker Congo Red, non-fluorescing dye whose differential absorptionspectra are used for selective detection of amyloid fibrils. As seen in Fig. 1A, increasing theconcentration of fibrillar lysozyme leads to progressive decrease of 3q absorption, with clearisosbestic point. Quantitative characteristics of the dye binding to protein aggregates ( Ka–association constant, n – binding stoichiometry, α – the difference of extinction coefficients inthe bound and free states) were estimated by analysing the isotherm of 3q binding tolysozyme fibrils in terms of Langmuir adsorption model (Fig. 1B). The recovered parametersare close to those reported for existing amyloid-sensitive chromophores.0.10NC3q0.05NCCH 2CH 2N(CH 3) 3Lysozyme amyloidCNClO4concentration, M0.00244563-0.057892104-0.10116350 400 450 500 550 600 650H 2CNWavelength, nmABFig.1. Differential absorption spectra of 3q (A) and the isotherm of 3q binding to fibrillarlysozyme (B).To conclude, spectral behavior of 3q as one representative of tricyanovinylarylamine dyesallowed us to recommend these compounds for testing as possible amyloid markers similar toCongo Red.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).- 34 -A 5020.100.080.060.040.020.00K a= 0.13 M -1n = 0.5 mol/mol = -0.0028 M -1 cm -10 20 40 60 80 100 120Protein concentration,