Наукові роботи. Факультет радіофізики, біомедичної електроніки та комп’ютерних систем
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Документ Spectral behavior of amyloid-specific dyes in protein-lipid systems. III. congo red interactions with native proteins(Харьковский Национальный Университет им. В.Н. Каразина, 2008) Kutsenko, O.K.; Trusova, V.M.; Gorbenko, G.P.; Dobrovolskaya, E.V.; Striha, O.A.; Derkach, R.V.A number of so-called conformational diseases (Parkinson's, Alzheimer's and Huntington's diseases, type II diabetes, spongiform encephalopathies, systemic amyloidosis) are associated with the deposition in various tissues highly-ordered protein aggregates (amyloid fibrils) that kill cells or prevent them from functioning properly. Amyloid fibrils are organized in a cross β-structure with a helical array of β-sheets, in which the long axis of the fibril is parallel to the long axis of the helix and is perpendicular to the β-strands Amyloid can be identified using a range of techniques: electron and atomic force microscopy, X-ray fibril diffraction, thioflavin T fluorescence, Congo Red (CR) birefringence or spectrophotometric assay. However, therapeutic detection of amyloid fibrils with CR test may be hampered by CR ability to form complexes with native proteins. In the present study we investigated CR binding to a series of native proteins – hemoglobin (Hb), cytochrome c (cyt c), ribonuclease A (RNase), human serum albumin (HSA). CR interaction with Hb and cyt c was followed by absorbance decrease and long wavelength shift of spectrum maximum in the case of Hb, indicating that native protein structure contains binding sites for CR. Association constant (Kb) and binding stoichiometry (n) recovered from the data analysis within the framework of Langmuir adsorption model were found to be: Kb=(2.1 ± 0.3)Ч105 M-1, n=3.3 ± 0.5 for Hb and Kb=(6.0 ± 0.9)Ч104 M-1, n=1.0 ± 0.3 for cyt c. The presence of lipid vesicles composed of phosphatidylcholine and cardiolipin did not exert influence on CR-Hb interactions. In contrast, association constant for CR-cyt c complexation markedly increased. This finding was interpreted in terms of cyt c unfolding at lipid-water interface coupled with exposure of additional CR binding sites on the protein surface. Formation of CR complexes with RNase and HSA was followed by the long-wavelength shift of absorption maxima. CR-HSA binding curves have Langmuir-like shape, whereas CR-RNase adsorption isotherms are slightly sigmoidal pointing to cooperative nature of the binding process. The binding parameters were estimated to be Kb=(1.3 ± 0.3)Ч104 M-1, n=2.3 ± 0.5 for HAS and Kb=(3.4 ± 0.3)Ч104 M-1, n=0.6 ± 0.1 and Hill parameter α= 1.1±0.2 for RNase.Документ Cytochrome C – cardiolipin interactions: extended lipid anchorage revisited(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Trusova, V.M.Resonance energy transfer (RET) from antrylvinyl-labeled (AV) lipids to the heme moiety of cytochrome c (cyt c) has been employed to assess the molecular level details of cyt c interactions with negatively charged lipid membranes composed of phosphatidylcholine (PC) and its mixture with 10 or 20 mol % of cardiolipin (CL). At the lowest ionic strength used here (20 mM) RET profiles from neutral (AV-PC) and anionic (AV-CL) donor were virtually indistinguishable, suggesting that the peculiarities of cyt c association with lipid membranes containing different donors are identical. In contrast, elevating ionic strength up to 40 and 60 mM resulted in expected decrease of energy transfer efficiency in the case of AV-PC containing liposomes, but not for those with AV-CL where RET exhibited an unexpected enhancement with increasing ionic strength. Monte Carlo analysis of the results obtained allowed us to attribute this untypical behavior to the transition of CL into extended lipid conformation. The revealed peculiarities of cyt c – CL interactions are of great interest not only from the viewpoint of regulating cyt c electron transfer and apoptotic propensities but also for elucidating the general mechanisms by which membrane functional activities can be modulated by protein-lipid interactions.