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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.
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.
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.
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.
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.
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.
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.
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.
Lipid bilayer modification induced by fibrillar lysozyme: fluorescence spectroscopy study
(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Kastorna, A.P.; Trusova, V.M.; Gorbenko, G.P.
The correlation between neurodegenerative diseases (Parkinson’s, Alzheimer’s diseases), type II diabetes, systemic amyloidosis, etc. and deposition of protein aggregates in brain and other tissues has long been established. A growing body of evidence has demonstrated that binding of amyloid proteins to the membrane may underlie their cytotoxic effect. It was shown that amyloid toxicity arises primarily from a soluble oligomeric form (pre-fibrillar aggregates) of the peptide rather than amyloid monomers or mature fibrils. The molecular basis of the amyloid protein toxicity is not sufficiently clear and requires further investigation. In view of this, the present study has been undertaken to ascertain the effect of fibrillar aggregates of lysozyme on the structural and physical properties of model membranes (liposomes) composed of zwitterionic lipid phosphatidylcholine and its mixture with cholesterol (30 mol%). To this end, two fluorescent probes with different properties and bilayer location, pyrene and Laurdan, have been employed. Pyrene spectra have characteristic vibronic structure in the region of 370-400 nm. Relative intensities of vibronic transitions exhibit dependence on solvent polarity. Excited species of pyrene can interact with non-excited ones thus forming excited state dimers – excimers. Excimer-to-monomer fluorescence intensity ratio reflects the extent of pyrene excimerization, which depends mainly on the rate of monomer lateral diffusion in lipid bilayer, being a function of the density of lipid molecular packing. Analysis of pyrene emission spectra revealed the absence of any influence of fibrillar lysozyme on the structural state of bilayer acyl chain region. Laurdan is an amphiphilic fluorescent probe, whose emission spectra are sensitive to the environmental polarity (hydration level). In the solvents of high polarity, Laurdan shows a considerable shift of its emission spectrum to longer wavelengths due to the dipolar relaxation processes. The changes in the emission spectrum of Laurdan were characterized by the generalized polarization value (GP). In all types of liposomes increasing concentration of fibrillar lysozyme resulted in the increment of GP, suggesting that amyloid fibrils cause the decrease in the lipid bilayer polarity.
Lipid-mediated lysozyme aggregation: furster resonance energy transfer study
(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Trusova, V.M.
Aggregation of proteins into insoluble complexes is intimately linked to pathogenesis of several neurodegenerative diseases. Protein aggregation is commonly regarded as nonspecific coagulation of incompletely folded or partially denaturated polypeptides, driven by interaction between the exposed hydrophobic patches. Accumulating evidence indicates that protein self-association can be induced by protein-lipid interactions. The present study addresses a problem of aggregation behavior of lysozyme (Lz) bound to model membranes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) (8:2). The formation of Lz assemblies in lipid environment has been monitored by measuring steady-state resonance energy transfer (FRET). Several donor-acceptor pairs have been employed: tryptophan (Trp) – pyrene, pyrene – fluorescein 5’-isothiocyanate (FITC) and FITC – rhodamine-isothiocyanate (RITC). Fluorescence spectra of pyrene maleimide-labelled Lz (Lz-PM) recorded at different lipid-to-protein molar ratios (L:P) with excitation wavelength of 296 nm were featured by three bands corresponding to Trp, pyrene monomer and excimer emission. Analysis of the shape of emission spectra showed that the ratio of PM to Trp intensity rises with increasing Lz-PM concentration and decreasing L:P values from 379 to 77. This effect is most probably to arise from the enhanced FRET between Trp and pyrene. The finding that the magnitude of this effect depends on protein concentration suggests that FRET enhancement is caused by the formation of protein aggregates. The same result was obtained with Lz- attached pyrene as donor and Lz-attached FITC as acceptor – the efficiency of energy transfer increased with increasing total protein concentration and decreasing L:P. Notably, the most pronounced increase of energy transfer efficiency was observed at surface coverage ca. 38 lipids per one protein molecule suggesting that this L:P value is critical for formation of Lz self-associates. The assumption that Lz forms aggregates in membrane environment is also corroborated by the quantitative analysis of FRET between FITC and RITC. The distance between FITC and RITC was found to be ca. 8 nm which exceeds the dimensions of Lz molecule by 2-2.5 times, lending additional support to the idea about Lz self- association in lipid surroundings.
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.
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.
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.
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.
Quantitative analysis of the benzanthrone aminoderivative binding to amyloid fibrils of lysozyme
(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Vus, K.O.; Trusova, V.M.; Gorbenko, G.P.; Kirilova, E.; Kirilov, G.; Kalnina, I.
The accumulation of amyloid fibrils in different tissues is associated with a number of neurodegenerative diseases. Despite a huge variety of amyloid-specific probes, all of them suffer from many drawbacks, highlighting the necessity of searching for more preferable dyes. In the present work, the potential of new fluorescent probe AM3 for selective detection of fibrillar protein aggregates, formed from lysozyme, has been evaluated. To quantify the affinity of this dye for amyloid fibrils, the isotherms of dye binding to the fibrillar lysozyme have been derived from fluorimetric titration. Parameters of the dye-protein complexation: association constant, molar fluorescence and binding stoichiometry, calculated from the Langmuir adsorption model, revealed that AM3 interacts strongly with protein insoluble aggregates. High values of the binding parameters make AM3 an alternative to a widely-used amyloid-specific probe Thioflavin T. We also investigated the effects of polarity and viscosity on AM3 fluorescence properties. The binding of AM3 to the protein hydrophobic cavities has been followed by red shift of the dye emission spectra, which can be explained by H-bonding between proton-donating groups of the protein and carbonyl moiety of the probe. Long-wavelength shift of emission maximum was observed also upon increasing the excitation wavelength. This finding suggests that reorientation time of solvent molecules is higher, than the dye fluorescence lifetime. Fluorescence anisotropy studies revealed slow rotation diffusion of the probe, bound to amyloid fibrils being indicative of high viscosity of AM3 microenvironment. The observed photophysical properties of the new aminobenzanthrone derivative make AM3 a perspective probe for basic research and medical diagnostics.
Resonance energy transfer study of hemoglobin binding to model lipid membranes
(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Kutsenko, O.K.; Gorbenko, G.P.; Trusova, V.M.
In the present study fluorescence resonance energy transfer (FRET) technique was employed to obtain the information about the structure of hemoglobin (Hb) complexes with model lipid membranes of different composition. For this purpose three membrane probes, 3-methoxybenzanthrone (MBA), 4- dimethylaminochalcone (DMC) and 6-propionyl-2-dimethylaminonaphthalene (Prodan) were assessed as possible donors for heme moiety of the protein. Model membranes were composed of zwitterionic lipid phosphatidylcholine (PC), anionic lipid cardiolipin (CL) and cholesterol (Chol). FRET measurements were interpreted in terms of the model of energy transfer in two-dimensional systems proposed by Fung and Stryer and further extended by Davenport et al. No FRET was observed between Prodan and Hb because Prodan under the employed experimental conditions was not distributed into the lipid bilayer. In the case of DMC, Hb-induced oxidative processes in the lipid phase hampered the estimation of Hb location in a lipid bilayer. Therefore, structural analysis of Hb-lipid complexes was carried out using MBA as a donor. First, the donor quantum yield, Fцrster radii and fluorescence anisotropy of the probes have been measured. Second, the amount of Hb bound to model membranes was estimated in terms of the lattice models of large ligand adsorption to lipid bilayers allowing for the possibility of protein insertion into membrane interior. Finally, the distance from acceptor plane to the bilayer center and the depth of Hb penetration into lipid bilayer were calculated. It was assumed that protein binds to membranes in the form of dimers and penetrates into the membrane interior. In neutral liposomes Hb penetrates only to the depth of lipid headgroups. The observed higher extent of Hb penetration into Chol containing bilayer as compared to PC liposomes may be a consequence of specific Hb-Chol interaction. In the case of PC/CL liposomes Hb was found to insert in the non-polar membrane region. Taking into account the possibility of forming the inverted hexagonal structures in the presence of CL, it cannot be excluded that Hb being entrapped in such structures, translocates through the membrane. If this phenomenon takes place, deeper Hb penetration into lipid bilayer might be expected. The obtained results can be useful for exact characterization of Hb binding to the membranes.
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.
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.