Implications of albumin in cell culture media on the biological action of vanadates(V).

In this article, the implications of binding competition of vanadates(V) with dodecyl sulfates for bovine serum albumin on cytotoxicity of vanadium(V) species against prostate cancer cells have been investigated. The pH- and SDS-dependent vanadate(V)-BSA interactions were observed. At pH 5, there is only one site capable of binding ten vanadates(V) ions (logK(ITC)1 = 4.96 ± 0.06; ΔH(ITC)1 = -1.04 ± 0.03 kcal mol-1), whereas at pH 7 two distinctive binding sites on protein were found, saturated with two and seven V(V) ions, respectively (logK(ITC)1 = 6.11 ± 0.06; ΔH(ITC)1 = 0.78 ± 0.12 kcal mol-1; logK(ITC)2 = 4.80 ± 0.02; ΔH(ITC)2 = - 4.95 ± 0.14 kcal mol-1). SDS influences the stoichiometry and the stability of the resulting V(V)-BSA complexes. Finally, the cytotoxicity of vanadates(V) against prostate cancer cells (PC3 line) was examined in the presence and absence of SDS in the culture medium. In the case of a 24-h incubation with 100 µM vanadate(V), a ca. 20 % reduction in viability of PC3 cells was observed in the presence of SDS. However, in other considered cases (various concentrations and time of incubation) SDS does not affect the dose-dependent action of vanadates(V) on the investigated prostate cancer cells.

Investigation of hexacyanoferrate(II)/(III) charge-dependent interactions with bovine and human serum albumins.

In the present paper, the binding interactions of highly negative-charged ions, namely hexacyanoferrates(II/III), i.e. [Fe(CN)6]4- and [Fe(CN)6]3- with bovine and human serum albumins (BSA and HSA, respectively) have been studied for the first time in an aqueous solution (10 mM cacodylate buffer of pH 7.0) using steady-state fluorescence spectroscopy, isothermal titration calorimetry, and CD spectroscopy supported by molecular dynamics-based computational approaches. The Stern-Volmer equation as well as its modifications suggested that hexacyanoferrates(II/III) effectively quenched the intrinsic fluorescence of the albumins through a static mechanism. The proteins under study possess only one binding site on the surface capable of binding one mole of hexacyanoferrates(II/III) ions per one mole of albumin (HSA or BSA). The formation of albumin complexes is an enthalpy-driven process (|ΔHITC| > |TΔSITC|). The strength of the interactions depends mainly on the type of albumin, and changes as follows: BSA-K3[Fe(CN)6] âˆ¼ BSA-K4[Fe(CN)6] > HSA-K3[Fe(CN)6] âˆ¼ HSA-K4[Fe(CN)6]. Finally, potential binding sites of bovine and human serum albumins have been investigated and discussed based on a competitive fluorescence displacement assay (with warfarin and ibuprofen as site markers) and molecular dynamics simulations.

Effect of Tetraphenylborate on Physicochemical Properties of Bovine Serum Albumin.

The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]-) have been investigated by a set of experimental methods (isothermal titration calorimetry, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy) and molecular dynamics-based computational approaches. Two sets of structurally distinctive binding sites in BSA were found under the experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K). The obtained results, supported by the competitive interactions experiments of SDS with [B(Ph)4]- for BSA, enabled us to find the potential binding sites in BSA. The first site is located in the subdomain I A of the protein and binds two [B(Ph)4]- ions (logK(ITC)1 = 7.09 ± 0.10; ΔG(ITC)1 = -9.67 ± 0.14 kcal mol-1; ΔH(ITC)1 = -3.14 ± 0.12 kcal mol-1; TΔS(ITC)1 = -6.53 kcal mol-1), whereas the second site is localized in the subdomain III A and binds five ions (logK(ITC)2 = 5.39 ± 0.06; ΔG(ITC)2 = -7.35 ± 0.09 kcal mol-1; ΔH(ITC)2 = 4.00 ± 0.14 kcal mol-1; TΔS(ITC)2 = 11.3 kcal mol-1). The formation of the {[B(Ph)4]-}-BSA complex results in an increase in the thermal stability of the alfa-helical content, correlating with the saturation of the particular BSA binding sites, thus hindering its thermal unfolding.

Fluorescence Quenching Studies on the Interactions between Chosen Fluoroquinolones and Selected Stable TEMPO and PROXYL Nitroxides.

The influence of the stable 2,2,6,6-tetramethylpiperidinyl-N-oxyl (TEMPO) nitroxide and its six C4-substituted derivatives, as well as two C3-substituted analogues of 2,2,5,5-tetramethylpyrrolidynyl-N-oxyl (PROXYL) nitroxide on the chosen fluoroquinolone antibiotics (marbofloxacin, ciprofloxacin, danofloxacin, norfloxacin, enrofloxacin, levofloxacin and ofloxacin), has been examined in aqueous solutions by UV absorption as well as steady-state and time-resolved fluorescence spectroscopies. The mechanism of fluorescence quenching has been specified and proved to be purely dynamic (collisional) for all the studied systems, which was additionally confirmed by temperature dependence experiments. Moreover, the selected quenching parameters-that is, Stern-Volmer quenching constants and bimolecular quenching rate constants-have been determined and explained. The possibility of electron transfer was ruled out, and the quenching was found to be diffusion-limited, being a result of the increase in non-radiative processes. Furthermore, as the chosen nitroxides affected the fluorescence of fluoroquinolone antibiotics in different ways, an influence of the structure and the type of substituents in the molecules of both fluoroquinolones and stable radicals on the quenching efficiency has been determined and discussed. Finally, the impact of the solvent's polarity on the values of bimolecular quenching rate constants has been explained. The significance of the project comes from many applications of nitroxides in chemistry, biology and industry.

On the Effect of pH, Temperature, and Surfactant Structure on Bovine Serum Albumin-Cationic/Anionic/Nonionic Surfactants Interactions in Cacodylate Buffer-Fluorescence Quenching Studies Supported by UV Spectrophotometry and CD Spectroscopy.

Due to the fact that surfactant molecules are known to alter the structure (and consequently the function) of a protein, protein-surfactant interactions are very important in the biological, pharmaceutical, and cosmetic industries. Although there are numerous studies on the interactions of albumins with surfactants, the investigations are often performed at fixed environmental conditions and limited to separate surface-active agents and consequently do not present an appropriate comparison between their different types and structures. In the present paper, the interactions between selected cationic, anionic, and nonionic surfactants, namely hexadecylpyridinium chloride (CPC), hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), polyethylene glycol sorbitan monolaurate, monopalmitate, and monooleate (TWEEN 20, TWEEN 40, and TWEEN 80, respectively) with bovine serum albumin (BSA) were studied qualitatively and quantitatively in an aqueous solution (10 mM cacodylate buffer; pH 5.0 and 7.0) by steady-state fluorescence spectroscopy supported by UV spectrophotometry and CD spectroscopy. Since in the case of all studied systems, the fluorescence intensity of BSA decreased regularly and significantly under the action of the surfactants added, the fluorescence quenching mechanism was analyzed thoroughly with the use of the Stern-Volmer equation (and its modification) and attributed to the formation of BSA-surfactant complexes. The binding efficiency and mode of interactions were evaluated among others by the determination, comparison, and discussion of the values of binding (association) constants of the newly formed complexes and the corresponding thermodynamic parameters (ΔG, ΔH, ΔS). Furthermore, the influence of the structure of the chosen surfactants (charge of hydrophilic head and length of hydrophobic chain) as well as different environmental conditions (pH, temperature) on the binding mode and the strength of the interaction has been investigated and elucidated.

Modification of DNA structure by reactive nitrogen species as a result of 2-methoxyestradiol-induced neuronal nitric oxide synthase uncoupling in metastatic osteosarcoma cells.

2-methoxyestradiol (2-ME) is a physiological anticancer compound, metabolite of 17ß-estradiol. Previously, our group evidenced that from mechanistic point of view one of anticancer mechanisms of action of 2-ME is specific induction and nuclear hijacking of neuronal nitric oxide synthase (nNOS), resulting in local generation of nitro-oxidative stress and finally, cancer cell death. The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME. We further achieved to identify the specific reactive nitrogen species involved in DNA-damaging mechanism of 2-ME. The study was performed using metastatic osteosarcoma 143B cells. We detected the release of biologically active (free) nitric oxide (•NO) with concurrent measurements of peroxynitrite (ONOO-) in real time in a single cell of 143B cell line by using •NO/ONOO- sensitive microsensors after stimulation with calcium ionophore. Detection of nitrogen dioxide (•NO2) and determination of chemical rate constants were carried out by a stopped-flow technique. The affinity of reactive nitrogen species toward the guanine base of DNA was evaluated by density functional theory calculations. Expression and localization of nuclear factor NF-kB was determined using imaging cytometry, while cell viability assay was evaluated by MTT assay. Herein, we presented that 2-ME triggers pro-apoptotic signalling cascade by increasing cellular reactive nitrogen species overproduction - a result of enzymatic uncoupling of increased nNOS protein levels. In particular, we proved that ONOO- and •NO2 directly formed from peroxynitrous acid (ONOOH) and/or by auto-oxidation of •NO, are inducers of DNA damage in anticancer mechanism of 2-ME. Specifically, the affinity of reactive nitrogen species toward the guanine base of DNA, evaluated by density functional theory calculations, decreased in the order: ONOOH > ONOO- > â€¢NO2 > â€¢NO. Therefore, we propose to consider the specific inducers of nNOS as an effective tool in the field of chemotherapy.

A Pentapeptide with Tyrosine Moiety as Fluorescent Chemosensor for Selective Nanomolar-Level Detection of Copper(II) Ions.

Herein, we have investigated principally with the use of UV and fluorescence (steady-state and time-resolved) spectroscopy the interactions between selected pentapeptides with tyrosine residue (EYHHQ, EHYHQ, EHHQY, and KYHHE) and various metal ions (Cu2+, Mn2+, Co2+, Ni2+, Zn2+, Cr3+, Cd2+, Ag+, Pb2+, Sr2+, Ba2+, Ca2+, Mg2+, Al3+, Fe2+, and Ga3+) in order to establish the relationship between the position of a tyrosine residue in the peptide sequence and the metal ion-binding properties. Among the peptides studied, EHYHQ was evaluated as an efficient and selective ligand for developing a chemosensor for the detection of copper(II) ions. While significant fluorescence emission quenching was observed for that peptide in the presence of Cu2+ cations, other metal cations used at the same and at considerably higher concentrations caused a negligible change of the fluorescence emission spectrum, indicating a high selectivity of EHYHQ for Cu2+ ions. Under optimum conditions, fluorescence intensity was inversely proportional to the concentration of Cu2+ ions. The limit of detection of Cu2+ ions with the use of EHYHQ was determined at the level of 26.6 nM. The binding stoichiometry of the complexes of the studied peptides with Cu2+ ions was evaluated spectrophotometrically and fluorimetrically (as in the case of EHYHQ confirmed by mass spectrometry) and found to be 1:2 (Cu2+-peptide) for all the investigated systems. Furthermore, the stability constant (K) values of these complexes were determined. The reversibility of the proposed Cu2+ ions sensor was confirmed, the pH range where the sensor acts was determined, while its analytical performance was compared with some other reported recently fluorescent sensors. The mechanism of the interactions between EHYHQ and Cu2+ was proposed on the basis of NMR spectroscopy investigations.

Dihydroxy-Substituted Coumarins as Fluorescent Probes for Nanomolar-Level Detection of the 4-Amino-TEMPO Spin Label.

This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM-a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH-the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).

Copper(II) complexation by fragment of central part of FBP28 protein from Mus musculus.

Steady-state and time-resolved fluorescence spectroscopy, UV spectrophotometry and isothermal titration calorimetry techniques were used to study the coordinating properties of the 17aa peptide fragment (D17) derived from the central part of the mouse formin binding protein (FBP28 with the PDB code: 1E0L) towards Cu2+ ions. All the measurements were run in the 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 6.0). Under experimental conditions the formation of the 1:1 complex of Cu2+ ions with D17 is an entropy-driven process. Cu2+ ions cause the static fluorescence quenching of the peptide studied through the formation of a non-fluorescent complex. Furthermore, the thermal stability of D17 was discussed based on the results obtained from differential scanning fluorimetry (nanoDSF) data.

The impact of environmental contamination on the generation of reactive oxygen and nitrogen species - Consequences for plants and humans.

Environmental contaminants, such as heavy metals, nanomaterials, and pesticides, induce the formation of reactive oxygen and nitrogen species (RONS). Plants interact closely with the atmosphere, water, and soil, and consequently RONS intensely affect their biochemistry. For the past 30 years researchers have thoroughly examined the role of RONS in plant organisms and oxidative modifications to cellular components. Hydrogen peroxide, superoxide anion, nitrogen(II) oxide, and hydroxyl radicals have been found to take part in many metabolic pathways. In this review the various aspects of the oxidative stress induced by environmental contamination are described based on an analysis of literature. The review reinforces the contention that RONS play a dual role, that is, both a deleterious and a beneficial one, in plants. Environmental contamination affects human health, also, and so we have additionally described the impact of RONS on the coupled human - environment system.

Oligomerization of 2-chloroallyl alcohol by 2-pyridinecarboxylate complex of chromium(III) - new highly active and selective catalyst.

The new 2-pyridinecarboxylate (2-pic) complex of chromium(III) has been designed and synthesized as a new highly active and selective oligomerization catalyst. The crystal structure of the new compound has been determined by X-ray diffraction. The composition and purity of [Cr(2-pic)2(OH2)2]NO3 have been confirmed by several spectroscopic methods and the elemental analysis. Furthermore, the new complex has been investigated towards its catalytic activity for the oligomerization of 2-chloro-2-propen-1-ol under the atmospheric pressure and at room temperature. It has turned out that the novel catalyst exhibits a very high catalytic activity. Consequently, [Cr(2-pic)2(OH2)2]NO3 belongs to a new generation of non-metallocene catalysts.

New type of highly active chromium(III) catalysts containing both organic cations and anions designed for polymerization of beta-olefin derivatives.

The new type of catalysts designed for the olefin derivatives polymerization has been synthetized. The novel catalysts are chromium(III) salt type complexes composed of both organic cation and anion, i.e. [Cr(dipic)2][Cr(bipy)(dipic)H2O]∙2H2O and [Cr(dipic)2]Hdmbipy∙2.5 H2O. The compositions of these complexes have been confirmed by a number of instrumental methods including NMR, IR, UV-Vis, MS and elemental analysis ones. Moreover, the crystal structures of these novel catalysts were determined and reported. Furthermore, the [Cr(dipic)2][Cr(bipy)(dipic)H2O]∙2H2O and [Cr(dipic)2]Hdmbipy∙2.5H2O complexes have been studied towards their catalytic activity, after the activation by MMAO (modified methylaluminoxane), in the case of 2-chloro-2-propen-1-ol polymerization at 21 °C and atmospheric pressure. It has been found that novel catalysts, [Cr(dipic)2][Cr(bipy)(dipic)H2O]∙2H2O and [Cr(dipic)2]Hdmbipy∙2.5 H2O, exhibit a very high catalytic activity in the process of the polymerization of the beta-olefin derivatives. The products of a such catalyzed polymerization are the poly(allyl alcohol) derivatives.

Antioxidant and Cytoprotective Activity of Oxydiacetate Complexes of Cobalt(II) and Nickel(II) with 1,10-Phenantroline and 2,2'-Bipyridine.

The antioxidant properties of oxydiacetate complexes of cobalt(II) and nickel(II) with 1,10-phenantroline and 2,2'-bipyridine have been investigated towards the superoxide radical using the nitro blue tetrazolium chloride (NBT) test and the cyclic voltammetry (CV). Moreover, the biological activity of the complexes under study has been investigated in the Human Dermal Fibroblasts adult (HDFa) cell line. In the first step, the cytotoxic and the antiproliferative activities of the complexes were examined. Subsequently, the cytoprotective properties of the complexes have been investigated in an oxidative stress conditions induced by H2O2.

Method for detection of hydrogen peroxide in HT22 cells.

We have proposed a new method which can be applied in assessing the intracellular production of hydrogen peroxide. Using this assay we have examined the hydrogen peroxide generation during the L-glutamate induced oxidative stress in the HT22 hippocampal cells. The detection of hydrogen peroxide is based on two crucial reagents cis-[Cr(C2O4)(pm)(OH2)2]+ (pm denotes pyridoxamine) and 2-ketobutyrate. The results obtained indicate that the presented method can be a promising tool to detect hydrogen peroxide in biological samples, particularly in cellular experimental models.

Characterization and cytotoxic effect of aqua-(2,2',2''-nitrilotriacetato)-oxo-vanadium salts on human osteosarcoma cells.

The use of protonated N-heterocyclic compound, i.e. 2,2'-bipyridinium cation, [bpyH+], enabled to obtain the new nitrilotriacetate oxidovanadium(IV) salt of the stoichiometry [bpyH][VO(nta)(H2O)]H2O. The X-ray measurements have revealed that the compound comprises the discrete mononuclear [VO(nta)(H2O)]- coordination ion that can be rarely found among other known compounds containing nitrilotriacetate oxidovanadium(IV) moieties. The antitumor activity of [bpyH][VO(nta)(H2O)]H2O and its phenanthroline analogue, [phenH][VO(nta)(H2O)](H2O)0.5, towards human osteosarcoma cell lines (MG-63 and HOS) has been assessed (the LDH and BrdU tests) and referred to cis-Pt(NH3)2Cl2 (used as a positive control). The compounds exert a stronger cytotoxic effect on MG-63 and HOS cells than in untransformed human osteoblast cell line. Thus, the [VO(nta)(H2O)]- containing coordination compounds can be considered as possible antitumor agents in the osteosarcoma model of bone-related cells in culture.

Simultaneous determination of thermodynamic and kinetic parameters of aminopolycarbonate complexes of cobalt(II) and nickel(II) based on isothermal titration calorimetry data.

The influence of the different side chain residues on the thermodynamic and kinetic parameters for complexation reactions of the Co2+ and Ni2+ ions has been investigated by using the isothermal titration calorimetry (ITC) technique supported by potentiometric titration data. The study was concerned with the 2 common tripodal aminocarboxylate ligands, namely, nitrilotriacetic acid and N-(2-hydroxyethyl) iminodiacetic acid. Calorimetric measurements (ITC) were run in the 2-(N-morpholino)ethanesulfonic acid hydrate (2-(N-morpholino) ethanesulfonic acid), piperazine-N,N'-bis(2-ethanesulfonic acid), and dimethylarsenic acid buffers (0.1 mol L-1 , pH 6) at 298.15 K. The quantification of the metal-buffer interactions and their incorporation into the ITC data analysis enabled to obtain the pH-independent and buffer-independent thermodynamic parameters (K, ΔG, ΔH, and ΔS) for the reactions under study. Furthermore, the kinITC method was applied to obtain kinetic information on complexation reactions from the ITC data. Correlations, based on kinetic and thermodynamic data, between the kinetics of formation of Co2+ and Ni2+ complexes and their thermodynamic stabilities are discussed.

The development of 1,3-diphenylisobenzofuran as a highly selective probe for the detection and quantitative determination of hydrogen peroxide.

1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H2O2 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196-3.941 mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8 µM, respectively. The kinetics of the reaction between DBBF and H2O2 was also studied.

Probing the binding of Cu(2+) ions to a fragment of the Aß(1-42) polypeptide using fluorescence spectroscopy, isothermal titration calorimetry and molecular dynamics simulations.

Steady-state and time-resolved fluorescence quenching measurements supported by isothermal titration calorimetry (ITC) and molecular dynamics simulations (MD), with the NMR-derived restraints, were used to investigate the interactions of Cu(2+) ions with a fragment of the Aß(1-42) polypeptide, Aß(5-16) with the following sequence: Ac-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-NH2, denoted as HZ1. The studies presented in this paper, when compared with our previous results (Makowska et al., Spectrochim. Acta A 153: 451-456), show that the affinity of the peptide to metal ions is conformation-dependent. All the measurements were carried out in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, pH6.0. The Stern-Volmer equations, along with spectroscopic observations, were used to determine the quenching and binding parameters. The obtained results unequivocally suggest that Cu(2+) ions quench the fluorescence of HZ1 only through a static quenching mechanism, in contrast to the fragment from the N-terminal part of the FPB28 protein, with sequence Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr- NH2 (D9) and its derivative with a single point mutation: Ac-Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr- NH2 (D9_M), where dynamic quenching occurred. The thermodynamic parameters (ΔITCH, ΔITCS) for the interactions between Cu(2+) ions and the HZ1 peptide were determined from the calorimetric data. The conditional thermodynamic parameters suggest that, under the experimental conditions, the formation of the Cu(2+)-HZ1 complex is both an enthalpy and entropy driven process.

Influence of Primary Ligands (ODA, TDA) on Physicochemical and Biological Properties of Oxidovanadium (IV) Complexes with Bipy and Phen as Auxiliary Ligands.

The influence of the oxydiacetate (ODA) and thiodiacetate (TDA) ligands on the physicochemical and biological properties of the oxidovanadium(IV) ternary complexes of the VO(L)(B) type, where L denotes ODA or TDA and B denotes 2,2'-bipyridine (bipy) or 1,10-phenanthroline (phen), has been investigated. The stability of the complexes in aqueous solutions, assessed based on the potentiometric titration method, increases in the following direction: VO(TDA)(bipy) < VO(ODA)(bipy) < VO(TDA)(phen) < VO(ODA)(phen). Furthermore, the influence of the TDA and ODA ligands on the antioxidant activity of the oxidovanadium(IV) complexes toward superoxide free radical (O2•-), 2,2'-azinobis(3-ethylbenzothiazoline-6 sulfonic acid) cation radical (ABTS+•) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) has been examined and discussed. The reactivity of the complexes toward O2•- increases in the following direction: VO(TDA)(phen) < VO(TDA)(bipy) ≈ VO(ODA)(bipy) < VO(ODA)(phen). The antioxidant activity against ABTS+• and DPPH• free radicals is higher for phen complexes, whereas the thiodiacetate complexes are more reactive than are the corresponding oxydiacetate ones. Finally, herein, the cytoprotective activity of the complexes against the oxidative damage generated exogenously by hydrogen peroxide in the hippocampal neuronal HT22 cell line (the MTT and LDH tests) is reported. In a low concentration (1 µM), the cytoprotective action of thiodiacetate complexes is much higher than that of the corresponding oxydiacetate complexes. However, in the higher concentration range (10 and 100 µM), the antioxidant activity of the complexes is overcompensated by their cytotoxic effect.

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry.

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu(2+) with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15K in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu(2+) ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu(2+) ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu(2+) ions are discussed.