Факультет радіофізики, біомедичної електроніки та комп’ютерних систем
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Документ Fluorimetric study of interaction between europium coordination complexes and DNA(Харьковский Национальный Университет им. В.Н. Каразина, 2009) Kutsenko, O.K.; Trusova, V.M.; Gorbenko, G.P.; Limanskaya, L.A.; Deligeorgiev, T.; Vasilev, A.; Kaloyanova, S.; Lesev, N.Lanthanide coordination complexes have found numerous applications in a number of areas, including laser techniques, fluorescent analysis, biomedical assays. Likewise, they exhibit antitumor properties. Eu(III) tris-β-diketonato complexes (EC) are newly synthesized compounds with high anticancer activity. Despite extensive studies, the detailed mechanism of their biological effects is far from being resolved. Examining the interactions between EC and biological molecules in model systems is essential for deeper understanding of the mechanisms behind their biological activity. In the present work we employed fluorescent probe acridine orange (AO) to investigate EC-DNA interaction. AO-DNA binding was followed by the marked fluorescence increase detected at 530 nm. EC addition suppressed this fluorescent changes. EC were found to differ in their ability to modify AO-DNA interactions. EC4 and EC6 have demonstrated the most pronounced effect on AO-DNA binding. AO-DNA complexation occurs predominantly via intercalation mode. EC are large planar structures, whose DNA intercalating ability was reported to increase with the planarity of ligands. It seems likely that AO and EC can compete for the binding sites on DNA molecule.Документ Intrinsic fluorescence of lysozyme in model protein-lipid systems(Харьковский Национальный Университет им. В.Н. Каразина, 2009) Trusova, V.M.Fluorescence spectroscopy is one of the most powerful tools providing new insights into the structural, dynamic and functional behavior of biological macromolecules, being particularly useful in investigating the molecular details of protein-lipid association. Complete and accurate information about the conformational dynamics of protein molecules can be obtained using tryptophan (Trp) residues as intrinsic fluorescence probes. The fluorescence of indole chromophore is extremely sensitive to environment making it an ideal choice for reporting protein conformational transitions upon membrane interactions. Hen egg white lysozyme (Lz) is a multi-tryptophan protein which is extensively used in elucidating fundamental aspects of protein-lipid interactions. The main emitters responsible for 80% of lysozyme fluorescence are Trp62 and Trp108. The present study was undertaken to ascertain the alterations in lysozyme structural state upon association with model membranes composed of zwitterionic lipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and anionic lipid 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphoglycerol (POPG). Fluorescence lifetime measurements showed that intensity-averaged lifetime (<τ>) of Trp residues in lysozyme decreased upon the protein binding to model membranes. Furthermore, <τ> reduction from 1.94 to 1.74 ns was observed at decreasing lipid-to-protein molar ratio (L:P) from 1130 to 120. It was suggested that Trp specific interactions with certain amino acid residues in its surroundings is the main factor responsible for the recovered decrease in tryptophan lifetime and the observed contradictions between lifetime, quenching and steady-state experiments. Since Lz is a stable protein whose conformation is reported to change insignificantly upon the formation of protein-lipid contacts, it can be assumed that the processes behind the drop in <τ> involve Lz self-association in membrane-bound state. Trp62 and Trp108 are located in the protein active site which reportedly participates in Lz aggregation. Moreover, Cys76-Cys94 disulfide bridge capable of efficient quenching of Trp fluorescence and reducing the lifetime of protein fluorophores, also resides in the active site cleft. Thus, it may be supposed that interactions between Trp62 and Trp108 of one Lz monomeric molecule with disulfide bridge of another monomeric molecule during the protein aggregation result in reduction of <τ> values. The <τ> dependence on L:P can be explained by the fact that lysozyme self-association is apparently coverage-dependent process controlled by both electrostatic and hydrophobic protein-lipid interactions. Additional arguments in favor of the assumption on Lz aggregation come from the time- resolved anisotropy measurements. Lysozyme rotational correlation time which reflects the motion of the whole protein molecule, was found to exhibit twofold increase at increasing L:P values. The recovered membrane ability to modulate Lz aggregation behavior may largely determine the bactericidal and amyloidogenic propensities of this protein.Документ Liposomal formulations of antitumor drugs. II. effect of lipid compositions on membrane interactions of europium coordination complexes(Харьковский Национальный Университет им. В.Н. Каразина, 2009) Yudintsev, A.V.; Trusova, V.M.; Gorbenko, G.P.; Deligeorgiev, T.; Vasilev, A.; Gadjev, N.Currently there is a growing interest in screening of new drugs, capable of destroying cancer cells effectively, without damaging health tissues. In this context the potential of liposomes as a drug carrier system is extensively investigated [1-3]. Liposomes are nanosize particles in which lipid bilayer encloses an aqueous internal compartment. Size, charge and surface properties of liposomes can be easily changed simply by adding new ingredients to the lipid mixture before liposome preparation or by variation of preparation techniques. Another important feature is that lipid vesicles can entrap both hydrophilic and hydrophobic pharmaceutical agents. Liposome delivery systems can enhance drug solubility, reduce toxicity associated with free anticancer drugs and improve stability of the drug by protecting the compound from chemical degradation or transformation. However, the therapeutic and toxic effects of drug are strongly determined by the degree or efficiency of its loading into the liposomes. For this reason, while using liposomes as delivery systems for hydrophobic drugs, it is necessary to know the character of a drug effect on the structure and physicochemical properties of a lipid bilayer. The aim of this work was to investigate the effect of lipid composition on membrane interactions of europium coordination complexes, V3 and V4, the potential antineoplastic drugs. Liposomes were formed by egg yolk phosphatidylcholine (PC) and its mixture with cardiolipin (CL) and cetyltrimethylammonium bromide (CTAB). The membrane-partitioning properties of the investigated drugs were evaluated using the equilibrium dialysis technique in combination with absorption spectroscopy. To gain insight into the drug influence on physical parameters and molecular organization of lipid bilayer, two fluorescent probes have been employed, viz. pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH). It was found that inclusion of anionic lipid cardiolipin and cationic detergent CTAB into PC bilayer gives rise to decrease of the drugs partition coefficients. The drug incorporation into liposomal membrane is accompanied by the alterations of pyrene spectral parameters and DPH anisotropy. The observed effects suggest that the influence of europium compounds on bilayer structural state can be modulated by CL and CTAB.Документ Liposomal formulations of antitumor DRUGS. I. cholesterol effect on membrane interactions of europium coordination complexes(Харьковский Национальный Университет им. В.Н. Каразина, 2008) Yudintsev, A.V.; Trusova, V.M.; Gorbenko, G.P.; Deligeorgiev, T.; Vasilev, A.; Gadjev, N.Among a wide variety of drug nanocarriers developed to date, liposome-based delivery systems are particularly attractive due to their advantageous features such as biocompatibility, complete biodegradability, low toxicity, ability to carry both hydrophilic and lipophilic payloads and protect them from chemical degradation and transformation, increased therapeutic index of a drug, improved pharmacokinetic and pharmacodynamic profiles compared to free drugs, reduced side effects, etc. The efficiency of drug encapsulation is largely determined by its membrane-partitioning properties as well as physicochemical characteristics of the lipid vesicles. In the present study we concentrated our efforts on the pre-formulation studies of the two synthesized Eu(III) coordination complexes, V3 and V4, the potential anticancer drugs. More specifically, our goal was twofold: i) to characterize the membrane partition properties of these complexes, and ii) to assess how the lipid-associating ability of V3 and V4 depends on membrane structural state being varied by introducing the different amounts of cholesterol (Chol) into phosphatidylcholine (PC) lipid vesicles. To achieve this goal, several fluorescent probes including pyrene, 1,6-diphenyl-1,3,5-hexatriene (DPH), and 4-p-(dimethylaminostyryl)-1-dodecylpyridinium (DSP-12) have been employed. Partition coefficients of lanthanides determined using the equilibrium dialysis technique proved to depend on the amount of Chol content. Formation of drug-lipid complexes was found to affect pyrene excimerization and DSP-12 spectral properties but exerted no influence on pyrene vibronic structure and DPH anisotropy. Membrane composition was shown to have an impact on the spectral responses of the probes in drug-lipid systems. This finding was interpreted as arising from the sterol condensing effect on the structural state of the lipid bilayer.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 Interaction of novel benzanthrone derivative with amyloid lysozyme(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Vus, K.O.; Trusova, V.M.; Gorbenko, G.P.; Zhytniakivska, O.A.; Kirilova, E.; Kirilov, G.; Kalnina, I.A novel benzanthrone derivative AM18 was investigated with respect to its photophysical properties when bound to native, oligomeric and fibrillar hen egg white lysozyme. As shown by fluorimetric titration AM18 is more sensitive to pathogenic protein aggregates than Thioflavin T, however has no ability to differentiate between mature and immature lysozyme fibrils. The recovered affinity and fluorescence response of the novel probe to amyloid protein appeared to be similar to those of recently developed amyloid lysozyme-sensitive dyes like e. g. Nile Red and cyanine dye 7515. Despite the high increase of the probe emission in the presence of amyloid lysozyme compared to its fluorescence in buffer, the minimal amount that could be detected by 1 μM AM18 was 10 times lower for amyloid-native protein solutions due to high affinity of the dye for lysozyme monomers. In general, because of high quantum yields and “signal-to-noise” ratios in the presence of pathogenic protein aggregates AM18 appeared to be an effective tool for amyloid detection and characterization in vitro, being however unable to detect pathogenic protein aggregates in vivo like e.g. recently reported p-FTAA because of the sensitivity to lipids. Compared to previously reported AM3 a novel dye showed 2-fold lower “signal-to- noise” ratio in the presence of fibrillar lysozyme, and 2 fold lower blue shift of emission maximum. This tendency was explained in terms of decreased charge transfer from the donor to acceptor groupes of AM18 compared to AM3. Finally, as concluded from the comparison of AM18 and previously studied benzanthrone derivatives, the 5 nm – red edge excitation shift of AM18 is indicative of its possible binding to fibril “deep cavities”, containing no water. High anisotropy values of amyloid-bound dye led us to conclusion that the enhanced fluorescence of the probe is associated with the decrease of the rotational motion of the amino-substitute about the benzanthrone unit. This is a sign of AM18 behaviour as a molecular rotor.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 Hemoglobin binding to phospholipid membranes as revealed by pyrene fluorescence study(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Kutsenko, O.K.; Gorbenko, G.P.; Trusova, V.M.In this work hemoglobin (Hb) association with lipid bilayers was investigated using fluorescent probe pyrene. Model membranes were prepared from zwitterionic lipid phosphatidylcholine (PC), anionic lipid phosphatidylglycerol (PG) and cholesterol (Chol). Hb-lipid binding was followed by the pyrene fluorescence quenching. Hb-induced decrease of pyrene monomer fluorescence was followed by the increase of relative intensities of vibronic bands. Presumably, Hb penetration into the bilayer increases the space between neighbouring lipids and promotes water penetration into the membrane core. Pyrene excimer emission quenching was interpreted in terms of resonance energy transfer. The greatest depth of Hb penetration into the lipid bilayer was observed in PC vesicles. In Chol-containing liposomes sterol condensing effect prevents deep protein penetration into the membrane. PG has an ability to stabilize lipid bilayers due to the ordered state of its lipid tails and H-bonding interactions between lipid molecules. This also can prevent Hb access to the inner membrane regions.Документ Influence of oligomeric and fibrillar lysozyme on physical properties of model membranes(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Kastorna, A.P.; Trusova, V.M.; Gorbenko, G.P.A pathological hallmark of more than 20 human diseases including Alzheimer’s disease, Parkinson’s disease, type II diabetes is the deposition in organs and tissues of insoluble highly ordered protein aggregates, called amyloid fibrils. It is becoming widely recognized that toxicity of amyloid species is related to their interactions with cell membranes. In the present study we focused our efforts on the examination of the influence of amyloid fibrils and their precursors (oligomeric aggregates) of lysozyme on the structural and physical properties of the model membranes composed of phosphatidylcholine and its mixture with cholesterol. For evaluating the extent of lipid bilayer modifications, we used fluorescence spectroscopy technique. The results of pyrene excimerization measurements showed that amyloid protein reduces membrane fluidity. Analysis of Laurdan emission spectra revealed the ability of lysozyme aggregates to produce bilayer dehydration. The most pronounced membrane-modifying effects were observed in the case of oligomeric lysozyme. Significantly less influence of pathogenic protein aggregates on the physical properties of cholesterol-containing vesicles confirmed the hypothesis on the preventive role of cholesterol in amyloid-related diseases.