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 Chlorpromazine effect on lysozyme-lipid interactions(Харьковский Национальный Университет им. В.Н. Каразина, 2008) Trusova, V.M.Despite considerable research efforts, the molecular mechanisms of anaesthetic action still remain the matter of extensive debates. According to one viewpoint, anaesthetics alter the properties of lipid bilayer which, in turn, affects the functions of embedded membrane proteins. In contrast, protein-based theories of anaesthetic action postulate that the drugs modulate the functions of membrane proteins through direct association. To develop a unique conception of anaesthesia further in-depth investigations of drug-membrane interactions are strongly required. In the present work a well-known fluorescent probe pyrene has been employed to gain molecular insights into the interactions between amphipathic phenothiazine derivative chlorpromazine (CPZ) and model membranes composed of cationic globular protein lysozyme (Lz), and lipid vesicles prepared from zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in the molar ratios 19:1, 9:1 and 4:1. To give unambiguous interpretation of the drug effect on protein-lipid interactions, we first analyzed the changes in pyrene excimerization due to the formation of either CPZ-lipid or Lz-lipid complexes. Pyrene excimer-to-monomer intensity ratio (E/M), a parameter which reflects the alterations in membrane free volume, was found to decrease upon Lz or CPZ binding to the lipid vesicles. Apparently, embedment of the protein and drug molecules into the hydrophobic region of lipid bilayer gives rise to the increase in lipid packing, decrease in the rate of trans-gauche isomerization of the lipid acyl chains and, consequently, reduction of membrane free volume. At the next step of the study, we analysed the changes in the rate of pyrene excimerization upon Lz addition to drug-lipid mixtures. In CL-containing liposomes the presence of CPZ does not modify the magnitude and sign of protein effect on membrane free volume. This implies that CPZ is incapable of perturbing Lz structure and exerted no influence on the protein interactions with this kind of liposomes. In contrast, in PC vesicles E/M ratio appeared to increase upon lysozyme binding to CPZ-modified model membranes. This finding may be explained in terms of two possibilities: (i) CPZ induces the formation of the new Lz conformer whose interactions with lipid bilayers are accompanied by the increase in membrane free volume; (ii) CPZ imparts the positive charge to the lipid bilayer thereby preventing Lz penetration into hydrophobic membrane region. Interfacially-located protein molecules are likely to generate structural defects coupled with the increased bilayer free volume. The results presented here clearly demonstrate that membrane composition can modulate the drug action on lipid-protein interactions. The recovered difference between CPZ effect on Lz-lipid binding in PC and CL-containing bilayers provide support to the idea that membrane environment can stabilize certain protein conformations differing in their responsiveness to drug action.Документ Spectral behavior of novel benzanthrone probe in model membranes(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Zhytniakivska, O.A.; Kutsenko, O.K.; Trusova, V.M.; Gorbenko, G.P.; Kirilova, E.M.; Kirilov, G.K.; Kalnina, I.The present study was undertaken to evaluate the sensitivity of a newly synthesized benzanthrone dye to the changes in physicochemical properties of lipid bilayer. It was shown that the dye under study is non- emissive in buffer but exhibites strong fluorescence in lipid phase. Partitioning of AM15 into model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin and cholesterol was followed by significant increase of fluorescence quantum yield. Analysis of the partition coefficients showed that inclusion of cardiolipin and choleterol into phosphatidylcholine bilayer gives rise to the decrease of AM15 incorporation into lipid phase compared to the neat phosphatidylcholine membrane. It is assumed that AM15 resides in the hydrophobic bilater region, being oriented parallel to the lipid acyl chains.Документ Interaction of new fluorescent ict-dyes with lipid membranes(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Zhytnyakovskaya, O.A.; Kutsenko, O.K.; Trusova, V.M.; Gorbenko, G.P.; Deligeorgiev, T.; Kaloyanova, S.; Lesev, N.The present study was undertaken to evaluate the sensitivity of newly synthesized ICT dyes to the changes in physicochemical properties of lipid bilayer. Partitioning of ICT4 into lipid phase of the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin and cholesterol was followed by the decrease of fluorescence quantum yield and short- wavelength shift of emission maximum. On the contrary, ICT2 exhibited tenfold increase of the quantum yield upon interaction with liposomes, without any shift of the emission maximum. Analysis of the partition coefficients showed that inclusion of cardiolipin and choleterol into phosphatidylcholine bilayer gives rise to increase of the ICT2 incorporation into lipid phase compared to the neat phosphatidylcholine membrane.Документ Liposomal forms of new antitumor drugs based on europium chelates examined by P-terphenyl fluorescence quenching(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Limanskaya, L.A.; Trusova, V.M.; Gorbenko, G.P.; Deligeorgiev, T.; Vasilev, A.; Kaloianova, S.; Lesev, N.Europium chelates have been previously shown to possess pronounced cytotoxic activity. These compounds are of great interest for biomedical investigations and diagnostics, because their spectral characteristics are optimal for visualization of the occurred processes. Application of these pharmaceutical compounds in the free form is limited by their high toxicity and metabolic instability. In view of this, the development of the delivery systems for europium chelates becomes actual. Liposomes represent one of the most promising delivery systems, which allows to increase the efficiency of pharmacological agents. The use of liposomal formulations of antitumor drugs is currently in a focus of biomedical and biophysical research due to the following advantages: complete biocompatibility, ability to carry both lipophilic and hydrophilic compounds, protecting them from chemical degradation and transformation, decreased toxicity and increased therapeutic index of drug, etc. In the present work we explore the interaction between europium chelates (here referred to as V6 and V8) and model lipid membranes. Fluorescence intensity of membrane-incorporated probe p-terphenyl was found to decrease with enhancement of drugs concentration. The obtained results indicate that p-terphenyl fluorescence is quenched upon europium chelate incorporation into phosphatidylcholine liposomes. Quantitative characteristics of p-terphenyl fluorescence quenching by the drugs under consideration have been determined.Документ Partitioning of europium chelate into lipid bilayer as revealed by p-terphenyl and pyrene quenching(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Limanskaya, L.A.; Yudintsev, A.V.; Trusova, V.M.; Gorbenko, G.P.; Deligeorgiev, T.; Vasilev, A.; Kaloianova, S.; Lesev, N.Fluorescence quenching method is an effective tool for obtaining important information about different properties of biophysical and biochemical systems. In the present study quenching of fluorescent probes p-terphenyl and pyrene by europium chelate were observed in phosphatidylcholine liposomes. Europium chelates (EC) belong to a new class of potential antitumor drugs with high cytotoxic activity. These compounds are of particular interest for biomedical investigations and diagnostics, since their spectral characteristics are optimal for decrease of light scattering in biological patterns and background signal. However, the application of such drugs in a free form is limited by their high toxicity and metabolic instability. One efficient way to increase drug efficiency is based on using different drug delivery systems such as liposomes. Highly adaptable liposome-based nanocarriers currently attract increasing attention, because of their advantages, viz. complete biodegradability, ability to carry both hydrophilic and lipophilic payloads and protect them from chemical degradation and transformation, increased therapeutic index of drug, flexibility in coupling with targeting and imaging ligands, improved pharmacodynamic profiles compared to the free drugs, etc. The present study was focused on examination of lipid bilayer interactions of europium chelate (here referred to as V10). Fluorescence intensity of membrane- incorporated probes – pyrene and p-terphenyl – was found to decrease with increasing concentration of the drug, suggesting that V10 represents an effective quencher for these probes. This finding was explained by the drug penetration into hydrophobic membrane core, followed by the collision between V10 and probe molecules and subsequent fluorescence quenching. The acquired fluorescence quenching data were quantitatively interpreted in terms of the dynamic quenching model.