Binding Of Orange Research Articles

Orange G is a potential inhibitor of human insulin amyloid fibrillation and can be used as a probe to study mechanism of amyloid fibrillation and its inhibition

The extracellular insoluble deposits of highly ordered cross-β-structure-containing amyloid fibrils form the pathological basis for protein misfolding diseases. As amyloid fibrils are cytotoxic, inhibition of the process is a therapeutic strategy. Several small molecules have been identified and used as fibrillation inhibitors in the recent past. In this work, we investigate the effect of Orange G on insulin amyloid formation using fluorescence-based assays and negative-stain electron microscopy (EM). We show that Orange G effectively attenuates nucleation, thereby inhibiting amyloid fibrillation in a dose-dependent manner. Fluorescence quenching titrations of Orange G showed a reasonably strong binding affinity to native insulin. Binding isotherm measurements revealed the binding of Orange G to pre-formed insulin fibrils too, indicating that Orange G likely binds and stabilizes the mature fibrils and prevents the release of toxic oligomers which could be potential nuclei or templates for further fibrillation. Molecular docking of Orange G with native insulin and amyloid-like peptide structures were also carried out to analyse the contributing interactions and binding free energy. The findings of our study emphasize the use of Orange G as a molecular probe to identify and design inhibitors of amyloid fibrillation and to investigate the structural and toxic mechanisms underlying amyloid formation.

Transport of Molecular Cargo by Interaction with Virus‐Like Particle RNA

AbstractVirus‐like particles (VLPs) derived from Leviviridae virions contain substantial amounts of cellular and plasmid‐derived RNA. This encapsidated polynucleotide serves as a reservoir for the efficient binding of the intercalating dye thiazole orange (TO). Polyethylene glycol (PEG) molecules and oligopeptides of varying length, end‐functionalized with TO, were loaded into VLPs up to approximately 50 % of the mass of the capsid protein (hundreds to thousands of cargo molecules per particle, depending on size). The kinetics of TO–PEG binding included a significant entropic cost for the reptation of long chains through the capsid pores. Cargo molecules were released over periods of 20–120 hours following simple reversible first‐order kinetics in most cases. These observations define a simple general method for the noncovalent packaging, and subsequent release, of functional molecules inside nucleoprotein nanocages in a manner independent of modifications to the capsid protein.

Transport of Molecular Cargo by Interaction with Virus-Like Particle RNA.

Virus-like particles (VLPs) derived from Leviviridae virions contain substantial amounts of cellular and plasmid-derived RNA. This encapsidated polynucleotide serves as a reservoir for the efficient binding of the intercalating dye thiazole orange (TO). Polyethylene glycol (PEG) molecules and oligopeptides of varying length, end-functionalized with TO, were loaded into VLPs up to approximately 50 % of the mass of the capsid protein (hundreds to thousands of cargo molecules per particle, depending on size). The kinetics of TO-PEG binding included a significant entropic cost for the reptation of long chains through the capsid pores. Cargo molecules were released over periods of 20-120 hours following simple reversible first-order kinetics in most cases. These observations define a simple general method for the noncovalent packaging, and subsequent release, of functional molecules inside nucleoprotein nanocages in a manner independent of modifications to the capsid protein.

Reticulated platelets interfere with flow cytometric reticulocyte counts

As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis.

Thermochemically induced binding of methyl orange to polycations containing primary amine groups

AbstractThe thermochemical transformation of electrostatically formed complexes of methyl orange (MO) with polycations containing primary amine groups such as ammonium salts afforded new polymers with a high concentration of covalently bound 4‐N,N‐dimethylaminoazobenzene groups in the side chain. Poly(allylamine hydrochloride) and poly(β‐aminoethylene acrylamide hydrochloride) were employed as support polycations for MO. The transformation of sulfonate–ammonium ion pairs into sulfonamide bonds, via heating at an elevated temperature, was supported by the polymer properties before and after the thermal treatment. The polymer structure changes were monitored with elemental analysis, Fourier transform infrared, 1H NMR, and ultraviolet–visible absorption spectroscopy, and thermogravimetric analysis. The spacer length between the backbone and azobenzene structures used as side chains strongly influenced the polymer properties before and after the heat‐induced reaction. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5898–5908, 2006

Performance and mechanism in binding of Reactive Orange 16 to various types of sludge

Four types of waste sludge were collected from a drinking water treatment plant (waterworks sludge; WS), sewage treatment plant (sewage sludge; SS), anaerobic digestion (digested sludge; DS) and landfill site (landfill sludge; LS). These sludges were evaluated as biosorbents for the removal of Reactive Orange 16 (RO 16). The maximum sorption capacities of the four waste sludges estimated using the Langmuir equation at pH 2 were in order LS (159.0 ± 6.0 mg g −1) > SS (114.7 ± 4.7 mg g −1) > DS (86.8 ± 4.5 mg g −1) > WS (47.0 ± 5.8 mg g −1). However, the desorption efficiency of LS was insufficient (43.2%) compared with that of SS (90.0%), indicating that the SS has the potential to be regenerated as a biosorbent. From the potentiometric titration and FTIR study, the binding sites present in the SS were considered to be protonated amine groups (–NH 3 +), which play a role in the electrostatic interaction with the negatively charged sulfonate groups of RO 16.

Binding of methyl orange or ethyl orange dyes by some dioxolane copolymers: Synthesis of the copolymers and thermodynamics of the dye-copolymer interactions

The extent of binding of methyl orange or ethyl orange by (2-phenyl-1,3-dioxolane-4-yl) methyl methacrylate (PDMMA), 2-hydroxyethyl methacrylate (HEMA), and vinyl-pyrrolidone (VPy) copolymers has been investigated by the equilibrium dialysis method. The dialysis experiments have been carried out in a Tris (hydroxy methyl) aminomethane buffer (pH = 7) and at the temperatures of 15, 25, and 35°C. The PDMMA-co-HEMA and PDMMA-co-VPy copolymers have been prepared in the laboratory by using the related monomers in different ratios. The synthesized products were analyzed by Fourier Transform-infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR) spectroscopy, differential thermal analysis (DTA), and differential scanning calorimetry (DSC) techniques. The binding extent of the dyes by the copolymers was determined by ultraviolet (UV) absorbance measurements. The results indicate that the extent of binding is relatively higher for ethyl orange than that for methyl orange under identical conditions. The binding slightly decrease with increasing temperature, and it is accompanied with favorable free energy, and exothermic enthalpy change within the temperature range studied. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3355–3361, 2004

Ligand‐Induced Protein Transconformation Disclosed by Equilibrium Gel Filtration

Multiple binding of some sulfonic dyes with trypsin in acidic media was investigated by equilibrium gel filtration. The apparent dissociation constants for the binding of flavianate, orange II, and orange G were, respectively, 46, 6, and 61 µM, reflecting differences in the organic moieties of the dyes. The elution profiles displayed a negative correlation of the eluted volumes between the monophasic trough and the peak with saturation ratios. This correlation was attributed to the outcome of distinct tautomeric forms of trypsin induced by ligand, together with the role of the dextran polymer and self‐association of dye molecules. Protein transconformation was achieved after a lag time‐depending on the ligand structure and concentration inside the column.

Study of Inclusion Complexes of Cyclodextrins with Orange II

Inclusion reactions of α- and β-CD with Orange IIwere measured by means of a stopped-flow method and UV-spectra. The forward rate constants (k+), backward rateconstants (k-) and the equilibrium constants (K) for the inclusion reactions were determined. Theinclusion processes of CD with Orange II follow a one-step reaction mechanism. The value of the forward rate constantat neutral and alkaline conditions for Orange II binding to α-CD cavity was large. This suggests that thenaphthalene side is partially included in the final complex form. In the case of β-CD, only marginal differenceis shown.It is probable that inclusion covers the whole naphthalene group and part of the azo group as well,resulting ina small effect from the pH variation. The results show that the equilibrium constant at pH 7 for the binding ofOrange II to α-CD is the largest because the included part of Orange II matches the cavity of α-CD well.

Surface expression and functional characterization of α-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin

Factor V (FV) present in platelet alpha-granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, alpha-granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% +/- 4.7% of the total population and is referred to as convulxin and thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187-activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of alpha-granule FV and the hemostatic effectiveness of young platelets. (Blood. 2000;95:1694-1702)

Immobilization of a Water-Soluble Host Molecule by Polyion Complexation with a Polymer

Formation of a polyion complex between the cation of a calix[6]arene ammonium chloride, which has a long alkyl chain, and anionic chondroitin sulfate results in a precipitate in aqueous solution. After dissolution of the complex in an organic solvent, evaporation of this solution gives rise to a water-insoluble transparent film. The film is strong enough for handling and shows binding of Methyl Orange, indicating that it retains a fundamental function of the parent host molecule. Immobilization of an anionic sulfonated calix[6]arene can also be achieved by formation of a polyion complex with the cation of a poly(diallyldimethylammonium chloride). The film prepared from this complex exhibits the ability to incorporate Phenol Blue; this suggests that the binding site remains in the polyion complex film.

Binding of Fatty acid Anions to .BETA.-Conglycinin: Physical and Chemical Modification Studies on the Role of Protein Conformation and Positively Charged Groups as Binding Sites.

Binding of decanoate to soybean β-conglycinin has been studied using physically and chemically modified β-conglycinin. The role of the native conformation and the cationic groups as the binding sites and the location on the amino acid sequence have been investigated. The native conformation with a quaternary structure showed the highest number of bound ligand. None of the modified β-conglycinins binds decanoate quite as firmly as the intact β-conglycinin molecule. Competitive binding studies demonstrated that the binding of methyl orange and fatty acid to β-conglycinin occurred at the same cationic sites. The association of carboxylate anions with β-conglycinin involves electrostatic attraction of the carboxylate anions to protein cationic sites that lied close by the NH2-terminal together with the hydrophobic interactions between the fatty acid hydrocarbon tail and the nonpolar side chains of the protein. Studies with reduced and non-reduced β-conglycinins have provided additional insight into the binding mechanism. The disulfide bond formed between subunits reduces the flexibility of the β-conglycinin molecule, especially around the NH2-terminal segment, producing marked changes in the fatty acid binding characteristics of β-conglycinin. The strong fatty acid binding sites may be located in the cleft between the α and α' subunit contact regions.

ChemInform Abstract: A Conductance Study of the Binding of Methyl Orange, o‐Methyl Red and p‐Methyl Red Anions by β‐Cyclodextrin in Water

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A Conductance Study of the Binding of Methyl Orange, o‐Methyl Red and p‐Methyl Red Anions by β‐Cyclodextrin in Water

AbstractA conductance study concerning the binding of methyl orange (MO), o‐methyl red (o‐MR) and p‐methyl red (p‐MR) anions by β‐cyclodextrin (β‐CD) in water has been carried out at 25 °C. The observed molar conductivities (Λs) were found to decrease significantly for mole ratios of cyclodextrin/azo dye less than unity. A model involving 1:1 stoichiometry has been used to analyze the conductance data. The stability constant (K) and the molar conductivity (Λc) for each 1:1 inclusion complex were determined from the conductance data by using a nonlinear least squares curve fitting procedure. The binding sequence, based on the value of K at 25 °C, is found to be p‐MR &gt; MO &gt; o‐MR. The values of the ionic molar conductivities of the free and complexed anions at infinite dilution are reported.

Conductance study of the binding of methyl orange, o-methyl red and p-methyl red anions by α-cyclodextrin in water

A conductance study concerning the binding of methyl orange (MO, 4-[4-(dimethylamino)phenylaza]benzene-sulfonic acid), o-methyl red (o-MR, 2-[4-(dimethylamino)phenylaza]benzoic acid) and p-methyl red (p-MR) anions by α-cyclodextrin (α-CD) in water has been carried out at 25 °C. The observed molar conductances (Λs) were found to decrease significantly for mole ratios less than unity. A model involving 1 : 1 stoichiometry has been used to analyse the conductance data. The stability constant (K) and the molar conductance (Λc) for each 1 : 1 inclusion complex were determined from the conductance data by using a non-linear least-squares curve-fitting procedure. The binding sequence, based on the value of K at 25 °C, is found to be p-MR > MO > o-MR. The values of the ionic molar conductances of the complexed anions at infinite dilution are reported and the differences between such values and those of uncomplexed anions are in accord with the above binding sequence.

Temperature dependence of the binding of methyl orange by crosslinked poly(4-vinylpyridine)

Various crosslinked poly(4-vinylpyridines) (CP4VP) with different degrees of crosslinking were prepared by radical copolymerization of 4-vinylpyridine with N, N′-tetramethylenebisacrylamide as crosslinker. The binding abilities of these crosslinked polymers were investigated at various temperatures in a buffer solution of pH 7. The first binding constant ( K 1) and thermodynamic parameters were evaluated from the equilibrium amounts of binding. The first binding constants showed bell-shaped curves when plotted against both the binding temperature and the degree of crosslinking. Lower temperature and higher degree of crosslinking at maximum binding in these bell-shaped curves were observed for this binding system when compared with those of the previously reported binding system (CP4VP/methyl organe) containing CP4VP prepared using N, N′-methylenebisacrylamide as crosslinker. The values of the enthalpy and entropy change increased on increasing the degree of crosslinking and decreasing the binding temperature, whereas the absolute magnitude of the free-energy change was not increased. These results could be accounted for in terms of the temperature dependence of the hole size of CP4VP in addition to the hydrophobic interactions in the binding process.

Binding of Acid and Basic Dyes by Wool Keratin Derivatives

Wool keratin derivatives such as S-carboxymethylated, S-cyanoethylated, S-sulfo nated, and regenerated keratin derivatives have been prepared to study the binding of acid and basic dyes (methyl orange, chrome violet, and acridine orange) by the derivatives in aqueous solution. The first binding constants and the thermodynamic parameters accompanying the binding were evaluated. The first binding constants of the proteins for the dyes were on the order of 105-106, which is similar to or higher than that of serum albumin. The favorable free energy of the binding of chrome violet and acridine orange observed for the formation of the dye-protein complex seems to be a result of a favorable enthalpy change rather than any favorable entropy change. The binding of methyl orange by the regenerated keratin derivative is accompanied by an entropy gain and an exothermic enthalpy change. The nature and phenomena of dyes binding with the keratin derivatives are discussed.

Binding of methyl orange and the hydrophobic fluorescent probe, 2‐p‐toluidinylnaphthalene‐6‐sulfonate, by 2‐dialkylaminoethyl methacrylate‐N‐vinyl‐2‐pyrrolidone and related copolymers

AbstractThe copolymers of N‐vinyl‐2‐pyrrolidone and 2‐dimethylaminoethyl methacrylate, 2‐diethylaminoethyl methacrylate, 2‐dimethylaminoethyl acrylate, or 2‐dimethylaminopropyl acrylamide have been prepared. Studies were made of the binding of a “binding probe,” methyl orange, by the copolymers in aqueous solution. The first binding constants accompanying the binding were evaluated. Furthermore, the intensity of fluorescence of a hydrophobic fluorescent probe, 2‐p‐toluidinylnaphthalene‐6‐sulfonate, in the presence of these polymers was investigated. The nature and phenomena of dye binding and hydrophobic fluorescent probe binding with the polymers are discussed.

Binding of butyl orange by poly(2‐dimethylaminoethyl methacrylate) and copolymers of 2‐dimethylaminoethyl methacrylate and N‐vinyl‐2‐pyrrolidone: Peculiar temperature dependence of the binding

AbstractThe temperature dependence of the binding of butyl orange by a homopolymer of 2‐dimethylaminoethyl methacrylate (DMAEMA) and copolymers of DMAEMA and N‐vinyl‐2‐pyrrolidone (VPy) has been examined at various pH's. The binding is very much dependent upon the temperature of the system, the pH of the binding medium, and the DMAEMA content in the polymer. In this case maximal binding is obtained at approximately 15–25° in the temperature range measured, although in most cases which have been examined, the degree of binding increases steadily with increasing temperature. This peculiar temperature dependence of the binding becomes more pronounced as the pH and the DMAEMA content are increased. The appearance of the peculiarity is discussed in terms of the pH‐induced conformational changes of the polymer and the hydrophobicity of the polymer.

Binding methyl orange and hydrophobic fluorescent probes by poly(2‐dimethylaminoethyl methacrylate) and copolymers of 2‐dimethylaminoethyl methacrylate and N‐vinyl‐2‐pyrrolidone: pH dependence of the binding and fluorescence intensity

AbstractThe pH dependence of the interaction of poly(2‐dimethylaminoethyl methacrylate) and copolymers of 2‐dimethylaminoethyl methacrylate and N‐vinyl‐2‐pyrrolidone with methyl orange, 2‐p‐toluidinylnaphthalene‐6‐sulfonate (TNS), and 1,6‐diphenyl‐1,3,5‐hexatriene (DHT) was studied by equilibrium dialysis and fluorescence measurements at pH's 7–10. The first binding constant accompanying the binding of methyl orange and TNS by the polymers, in particular the homopolymer, shows a maximum around pH 8 and maximal fluorescence intensity of TNS is obtained around pH 8.5 in the presence of the polymers. To elucidate these observations the pH‐induced conformational changes of the homopolymer were examined by potentiometric titration and viscosity measurements and the thermodynamic parameters that accompany the binding were calculated. The polymer was found to change from an extended coil at lower pH to a compact coil at higher pH. The electrostatic attraction between the sulfonate group of the small molecule and the protonated nitrogen atoms on the polymer is increased at lower pH and the hydrophobic interaction between the hydrophobic moieties of the polymer and the small molecule is enhanced at higher pH. The results obtained for the dye binding and fluorescence intensity were discussed in terms of the electrostatic and hydrophobic interactions.