Polyanion induced circular dichroism of Thioflavin T.

Thioflavin T (ThT) is amyloid specific fluorescence dye possessing the properties of molecular rotor. We have shown that Thioflavin T forms complexes with non-peptide polyanions heparin, polyadenylate and polystyrene sulphonate by means of absorption spectroscopy. In the presence of chiral polyanions - heparin and polyadenylate - induced optical activity of ThT occurs whereas interaction with achiral polystyrene sulphonate (PSS) does not lead to production of induced circular dichroism signal. The positively charged ThT forms centre for ordered binding of chiral polyanion. Similarly, complexation of structurally different chromophore 9-aminoacridine with polyanions has led to induction of optical activity only in the presence of chiral ones. We suggest that, primarily, the optical activity of environment plays important role in inducing optical activity of achiral compounds.

Water treatment using activated carbon supporting silver and magnetite.

Recent efforts in water purification have led to the development of novel materials whose unique properties can offer effective biocidal capabilities with greater ease of use and at lower cost. In this study, we introduce a novel procedure for the preparation of activated carbon (charcoal) composite in which magnetite and silver are incorporated (MCAG); we also describe the use of this material for the disinfection of surface water. The formation process of magnetic MCAG composite was studied using ultraviolet-visible spectroscopy. The results demonstrated the high sorption efficiency of AgNO3 to magnetic activated carbon. The antimicrobial capabilities of the prepared MCAG were examined and the results clearly demonstrate their inhibitory effect on total river water bacteria and on Pseudomonas koreensis and Bacillus mycoides cultures isolated from river water. The bacterial counts in river water samples were reduced by five orders of magnitude following 30 min of treatment using 1 g l⁻¹ of MCAG at room temperature. The removal of all bacteria from the surface water samples implies that the MCAG material would be a suitable disinfectant for such waters. In combination with its magnetic character, MCAG would be an excellent candidate for the simple ambulatory disinfection of surface water.

Determination of proton dissociation constants (pKa) of hydroxyl groups of 2,5-dihydroxy-1,4-benzoquinone (DHBQ) by UV-Vis, fluorescence and ATR-FTIR spectroscopy.

The dissociation constant is an important physicochemical parameter of amolecule. The protonation state of a molecule reflects its reactivity, solubility or ability to chemically interact with other molecules. In the present study, dissociation constants (pKa) values of 2,5-dihydroxy-1,4-benzoquinone (DHBQ) were determined by UV-Vis, fluorescence and ATR-FTIR spectroscopy at 25 °C. The resulting pKa values for DHBQ were 2.95 and 5.25. We have also experimentally found out that the monoanionic form (HBQ-) provides weak fluorescence in the pH range of about 3-6. This allowed us to determine not only the pKa in the ground but also the excited state of the molecule (pKa1* = 4.38 andpKa2* = 5.27).

Toxicological evaluation of fluorescent 11-mercaptoundecanoic gold nanoclusters as promising label-free bioimaging probes in different cancer cell lines.

Due to advancement in nanomaterials and increasing use of functionalized gold nanoclusters (AuNCs) in different biomedical applications, better understanding of their potential cytotoxicity is necessary. Interactions of ultra-small fluorescent AuNCs with mammalian cells remains up to this day poorly understood, therefore, cytotoxic evaluation of thoroughly characterized ca. 2.5 nm spherical water-soluble 11-mercaptoundecanoic acid coated AuNCs (AuNC@M) with diverse fluorescent properties in variety of mammalian cancer cell lines was performed. Cell viability was assessed by traditional MTT assay and xCELLigence real time cell analyzer. Cell apoptosis was evaluated via an Annexin V-FITC/propidium iodide (PI) assay. Confocal fluorescence imaging confirmed that tested AuNC@M entered live cells and were homogeneously distributed in their cytoplasm. The results suggested that the cytotoxicity of tested nanoclusters was very low, or near the control level at concentrations 0.1 and 0.5 mg/mL in the cell lines after 24 h exposition. The purity of tested AuNC@M had no relevant effect on cell viability and no differences were observed after 24 h in our study. The low toxicity toward cancer cells further strengthens our view that AuNC@M are promising label-free fluorescent probes for bio-labelling and bio-imaging, or they can even serve as platforms for antitumor drug delivery systems.

In vitro study of disodium cromoglicate as a novel effective hydrotrope solvent for hypericin utilisation in photodynamic therapy.

Hypericin (HY) is a naphthodianthrone that naturally occurs in Hypericum perforatum L. It is a promising photosensitiser used in photodynamic therapy for and diagnosis of oncological diseases. However, its hydrophobic character is an obstacle that has prevented its efficient use. The commonly used solvent, dimethyl sulfoxide (DMSO), is a controversial constituent of HY formulations and its use has been rejected by many researchers studying HY both in vitro and in vivo. In this study, we propose the utilisation of hydrotropy to solubilise HY in an aqueous environment. Cromolyn (DSCG) is a non-toxic, well-tolerated, antiallergic drug that has been employed in clinical practice since 1970, and in aqueous solution it acts as a hydrotrope. At a molecular ratio of 1:12,000 HY to DSCG, the compound is able to solubilise HY in aqueous environment. In an HT-29 cell suspension, DSCG (1.8 mmol L-1) considerably enhances the interaction between HY (150 nmol L-1) and HT-29 cells, which leads to an HY fluorescence emission increase with a half-time approximately 2 min compared to 29 min for samples that lack DSCG. Studies using HT-29 adenocarcinoma cells showed that DSCG at a given concentration significantly improved accumulation of HY within cells compared to DMSO (p < 0.05) despite the relative resistance of the HT-29 cell line to HY-PDT. Though no significant difference between total reactive oxygen species production was observed for photoactivated HY dissolved in DMSO and DSCG, significant singlet oxygen generation by photoactivated HY dissolved in a DSCG-containing water solution at the studied molecular ratio was confirmed. We also clarified that DSCG does not act as a scavenger of ABTS and galvinoxyl free radicals. The results from an MTT assay showed that DSCG also significantly enhanced the cytotoxicity of photoactivated HY compared to DMSO (p < 0.05). This study has demonstrated the ability of DSCG to act as a solvent of HY and enhance the effectiveness of HY-PDT compared to the commonly used organic solvent, DMSO.

Structural and thermodynamic behavior of cytochrome c assembled with glutathione-covered gold nanoparticles.

The conformational changes of horse heart ferricytochrome c (cyt c) after association of gold nanoparticles have been studied by electronic absorption spectroscopy and circular dichroism (CD). Our results show that the structural stability around the heme of complexed cyt c was increased successfully. Glutathione-layered gold nanoparticles caused a significant increase of the apparent pK values of the cyt c alkaline transition. Similarly, the heme crevice became more stable to heat after assembly of cyt c with gold nanoparticles. In contrast, gold nanoparticles weaken the overall thermal stability of the cyt c by decreasing the denaturation temperature estimated from far-UV CD measurements. Similar behavior has previously been reported for cyt c complexed with physiological redox partners as well as hydrophilic polyanions.

Effects of urea and acetic acid on the heme axial ligation structure of ferric myoglobin at very acidic pH.

The heme iron coordination of ferric myoglobin (Mb) in the presence of 9.0M urea and 8.0M acetic acid at acidic pH values has been probed by electronic absorption, magnetic circular dichroism and resonance Raman spectroscopic techniques. Unlike Mb at pH 2.0, where heme is not released from the protein despite the acid denaturation and the loss of the axial ligand, upon increasing the concentration of either urea or acetic acid, a spin state change is observed, and a novel, non-native six-coordinated high-spin species prevails, where heme is released from the protein.

Polyanion hydrophobicity and protein basicity affect protein stability in protein-polyanion complexes.

Stability of four dissimilar basic proteins (chymotrypsinogen A, ribonuclease A, cytochrome c, lysozyme) in the complex with four polyanions (heparin, poly(vinylsulfate), poly(4-styrene-sulfonate), Nafion) has been studied by differential scanning calorimetry. The polyanions were chosen because of their different charge density and hydrophobicity. Relative hydrophobicity of polyanions have been compared by three different parameters: (i) partition coefficient determined in octanol/water system, (ii) electrocapillary curves obtained by the method of controlled convection, and (iii) change in absorbance of small cationic amphiphilic molecule, aminoacridine, due to interaction with polyanion. Our results suggest that stability of proteins in the complex with polyanions negatively correlate with charge-related properties of the proteins such as isoelectric point and surface charge density and hydrophobicity of the polyanions.

A circular dichroism study of the stability of guanine quadruplexes of thrombin DNA aptamers at presence of K+ and Na+ ions.

The effect of K+ and Na+ on the properties of DNA aptamers that selective binds to fibrinogen (FIBRI) and heparin (HEPA) exosites of human alpha-thrombin was studied by circular dichroism (CD). The complexes of FIBRI-K+ were slightly more stable than HEPA-K+. However, lower stability was observed for HEPA-K+ at presence of Na+ in comparison with FIBRI-K+. The analysis of CD melting curves suggests differences in thermal stability of both aptamers at presence of K+. The melting temperatures (Tm) and changes in van't Hoff enthalpy for HEPA-K+ complexes were lower in comparison with those for FIBRI-K+. With increasing HEPA concentration the Tm value increased, but Tm did not change with increasing FIBRI concentration. This suggests formation of HEPA aggregates, while FIBRI aptamers are in monomeric form.

Excitation of triplet states of hypericin in water mediated by hydrotropic cromolyn sodium salt.

Hypericin (Hyp) is a hydrophobic pigment found in plants of the genus Hypericum which exhibits low levels of solubility in water. This work shows that the solubility of Hyp can be significantly increased through the addition of cromolyn disodium salt (DSCG). Performed studies using UV-VIS absorption and fluorescence spectroscopies demonstrate that Hyp remains in a predominantly biologically photodynamic active monomeric form in the presence of DSCG at concentrations ranging from 4.6×10-3 to 1.2×10-1mol·L-1. The low association constant between Hyp and DSCG (Ka=71.7±2M-1), and the polarity value of 0.3 determined for Hyp in a DSCG-water solution, lead to a suggestion that the monomerization of Hyp in aqueous solution can be explained as a result of the hydrotropic effect of DSCG. This hydrotropic effect is most likely a result of interactions between two relative rigid aromatic rings of DSCG and a delocalized charge on the surface of the Hyp molecule. The triplet-triplet (T-T) electronic transition observed in is Hyp in the presence of DSCG suggests a possible production of reactive oxygen species once Hyp is irradiated with visible light in a DSCG aqueous solution.

Irreversible thermal denaturation of elongation factor Ts from Thermus thermophilus effect of the residual structure and intermonomer disulfide bond.

The homodimeric wild-type elongation factor Ts, EF-Ts(wt), and its C190A mutant, EF-Ts(C190A), from Thermus thermophilus goes through thermal denaturation in a way consistent with a two state irreversible model with a relatively high activation energy, approximately 530 kJ/mol (Supplemental materials provides a list of 98 activation energies from 54 proteins in various solvent conditions). Removing the intermonomeric disulfide bond by substituting alanine for cysteine 190 affects the rate constant of the irreversible thermal transition. At physiological temperatures, the half-life of the native conformations was estimated to be approximately 21 days for wt and 1.3 days for C190A. Thermally denatured EF-Ts refolds into a molten-globule-like state as indicated by its native-like circular dichroism spectrum in the far UV region and the enhanced fluorescence of the hydrophobic probe, 1-anilinonaphtalene-8-sulphonate. The residual secondary structure observed in the thermally denatured state of EF-Ts at high temperatures affects its apparent temperature of thermal transition, T(trs), independent of the presence or absence of the intermonomeric disulfide bond. The effect of the GdmHCl concentration on the activation energy, E(a), and the temperature, T*, i.e., the temperature at which the rate of the irreversible step is 1 min(-1), indicates that the intermonomeric disulfide bond contributes to the irreversibility of thermal transition of EF-Ts.

The N-Terminal Region of the Ryanodine Receptor Affects Channel Activation.

Mutations in the cardiac ryanodine receptor (RyR2), the ion channel responsible for release of calcium ions from intracellular stores into cytoplasm, are the cause of several inherited cardiac arrhythmias. At the molecular level, disease symptoms can be mimicked by domain peptides from mutation-prone regions of RyR2 that bind to RyR2 and activate it. Here we show that the domain peptide DPcpvtN2, corresponding to the central helix of the N-terminal region of RyR2, activates the RyR2 channel. Structural modeling of interaction between DPcpvtN2 and the N-terminal region of RyR2 in the closed and open conformation provided three plausible structures of the complex. Only one of them could explain the dependence of RyR2 activity on concentration of DPcpvtN2. The structure of the complex was at odds with the previously proposed "domain switch" mechanism of competition between domain peptides and ryanodine receptor domains. Likewise, in structural models of the N-terminal region, the conformational changes induced by DPcpvtN2 binding were different from those induced by mutation of central helix amino acids. The activating effect of DPcpvtN2 binding and of mutations in the central helix could be explained by their similar effect on the transition energy between the closed and open conformation of RyR2.

Thermodynamic properties of nucleotide-free EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors of its GTPase activity.

The thermal transition of elongation factor EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors was studied by differential scanning calorimetry. The effectors of GTPase activity used were the antibiotic kirromycin and the cations Li(+), Na(+), K(+) and NH(4)(+) in the chloride form. The temperature of thermal denaturation and the cooperativity of the transition of nucleotide-free EF-Tu (EF-Tu(f)) in the presence of kirromycin are comparable with those of the EF-Tu x guanosine-5'-[beta,gamma-imido]triphosphate (GppNHp) form, indicating similar conformational states. Increased concentrations of Na(+) and K(+) stabilized EF-Tu(f) in a manner similar to GppNHp. NH(4)(+) decreased the transition temperature of EF-Tu(f) and Li(+) decreased both the temperature and the calorimetric enthalpy of the thermal transition of EF-Tu(f). In the presence of salts, binding of kirromycin had a stabilizing effect on EF-Tu(f). Correlation between the GTPase activity and thermodynamic characteristics of EF-Tu(f) induced by kirromycin in the absence or presence of the cations is discussed.

Microcalorimetric study of elongation factor Tu from Thermus thermophilus in nucleotide-free, GDP and GTP forms and in the presence of elongation factor Ts.

Elongation factor (EF) Tu undergoes profound nucleotide-dependent conformational changes in its functional cycle. The thermodynamic parameters of the different Thermus thermophilus EF-Tu forms, its domains I, II/III and III, were determined by microcalorimetry. Thermal transitions of the EF-Tu.GDP and EF-Tu.guanosine-5'-[beta,gamma-imido]triphosphate have a cooperative two-state character. Nucleotide removal affected the cooperativity of the thermal transition of EF-Tu. Microcalorimetric measurements of nucleotide-free EF-Tu and its separated domains showed that domains II/III have the main stabilizing role for the whole protein. Despite the fact that strong interactions between elongation factors Tu and Ts from T. thermophilus at 20 degrees C exist, the thermal transition of neither protein in the complex was significantly affected.

Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c.

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent pK(a) for the alkaline transition of cytochrome c. The change in pK(a) is comparable to the value when cytochrome c is complexed with cytochrome bc(1). Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met(80)-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3+/-0.2 to 66.9+/-0.2 degrees C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2+/-0.2 to 68.8+/-0.2 degrees C in the presence of polyglutamates with number of glutamate monomers n >or approximately equal 8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by approximately 42.5% with n>or approximately equal 8. The binding constant for the binding of octaglutamate (m.w. approximately 1000) to cyt c was found to be 1.15 x 10(5) M(-1) at pH 8.0 and low ionic strength. The results indicate that the polyglutamate (n>or approximately equal 8) is able to increase the stability of the methionine sulfur-heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.

The heme iron coordination of unfolded ferric and ferrous cytochrome c in neutral and acidic urea solutions. Spectroscopic and electrochemical studies.

The heme iron coordination of unfolded ferric and ferrous cytochrome c in the presence of 7-9 M urea at different pH values has been probed by several spectroscopic techniques including magnetic and natural circular dichroism (CD), electrochemistry, UV-visible (UV-vis) absorption and resonance Raman (RR). In 7-9 M urea at neutral pH, ferric cytochrome c is found to be predominantly a low spin bis-His-ligated heme center. In acidic 9 M urea solutions the UV-vis and near-infrared (NIR) magnetic circular dichroism (MCD) measurements have for the first time revealed the formation of a high spin His/H(2)O complex. The pK(a) for the neutral to acidic conversion is 5.2. In 9 M urea, ferrous cytochrome c is shown to retain its native ligation structure at pH 7. Formation of a five-coordinate high spin complex in equilibrium with the native form of ferrous cytochrome c takes place below the pK(a) 4.8. The formal redox potential of the His/H(2)O complex of cytochrome c in 9 M urea at pH 3 was estimated to be -0.13 V, ca. 100 mV more positive than E degrees ' estimated for the bis-His complex of cytochrome c in urea solution at pH 7.

Thermodynamics of a diffusional protein folding reaction.

The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB.

Preparation of CuO/ZnO nanocomposite and its application as a cysteine/homocysteine colorimetric and fluorescence detector.

Cysteine and homocysteine play a crucial role in many biological functions but abnormal levels of these amino acids may lead to various forms of pathogenesis. Therefore, selective and easy-to-use methods for the detection of cysteine and homocysteine are essential for the early diagnosis of developing diseases. In this paper we report on a rapid, straightforward and highly selective method for the detection of cysteine (Cys) and homocysteine (Hcy) which uses a CuO/ZnO nanocomposite as a dual colorimetric and fluorometric assay. The presence of Cys and Hcy in a solution of these nanorods (NRs) induces a change in its color from light blue to dark grey which is visible to the naked eye. This is accompanied by a blue shift in the absorption spectra from 725 nm to 650 nm and a decrease in the intensity of CuO/ZnO nanocomposite emission. These changes are ascribed to the reduction of Cu(II) to Cu(0), and the oxidation of cysteine (homocysteine) and subsequent formation of the disulfide bond. This novel assay method does not respond to any other amino-acid which is present in living organisms; therefore the selective determination of cysteine (homocysteine) with a lower analyte limit of 40 µM (4.8 µg mL(-1)) can be carried out in aqueous solutions without the need for any sophisticated instrumentation, fluorophore molecules or complicated procedures.

Interaction of cytochrome c with zinc oxide nanoparticles.

The influence of pH on the interaction between horse heart ferricytochrome c (cyt c) and zinc oxide nanoparticles (ZnO NPs) has been studied by a small angle scattering as well as UV-vis and FTIR spectroscopy. The observations showed that the optimal pH for the association of protein with nanoparticles is in pH range 5.0-8.0. Almost no significant change in structure and thermodynamic stability of cytochrome c after the association with 60 nm ZnO NPs was performed by UV-vis and by a circular dichroism spectroscopy.

Alkaline transition of horse heart cytochrome c in the presence of ZnO nanoparticles.

The effect of zinc oxide nanoparticles (ZnO NPs) on cytochrome c (cyt c) in alkaline pH was studied with absorption spectroscopy and UV circular dichroism (CD). Spectral data from UV-vis spectroscopy and circular dichroism indicate only small changes in the native structure of the protein at neutral pH after the interaction with ZnO nanoparticles. The stability around the heme crevice of cyt c and therefore the switch of the axial ligand Met80 to Lys which occurs in conditions of higher pH was proven following the interaction of cytochrome c with ZnO nanoparticles. The formation of cyt c-ZnO NPs complex based on electrostatic attraction was accompanied by a significant increase in the apparent pKa constant of the alkaline transition of cyt c.