Nature of the thiamin-binding protein from chicken egg yolk

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.

Vitamin A and thyroxine carrier proteins in chicken plasma Steady-state control of the plasma level of free retinol-binding protein and free thyroxine

1. 1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occuring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. 6. The thyroxine-binding proteins were found to be two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz.,prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.

Separation of 5-methylcytosine-rich DNA using immobilized antibody

Anti-(5-methylcytosine) antibodies were immobilized on glutaraldehyde-activated Indion 48-R, a polystyrene resin with amino groups. Immobilized antibody was very stable and could be used several times without any apparent change in the initial binding capacity of the antibody-matrix. Fractions of total DNA from various animal and plant sources were retained on this column and could be eluted quantitatively with 1.0 m NaCl. The bound fraction was further characterized for its 5-methylcytosine content by restriction enzyme digestion patterns.

Biosorption of copper and zinc using waste Aspergillus niger biomass

Nonliving waste biomass consisting of Aspergillus niger attached to wheat bran was used as a biosorbent for the removal of copper and zinc from aqueous solutions. Copper and zinc uptake by the biomass obeyed Langmuir isotherms. The binding capacity of the biomass for copper was found to be higher than that for zinc. The metal uptake, expressed in milligrams per gram of biomass, was found to be a function of: the initial metal concentration (with the uptake decreasing with increasing initial concentration), the biomass loading (with the uptake decreasing with increasing biomass loading) and pH (with the uptake increasing with increasing pH in the range of 1.5 and 6.0). The metal uptake was significantly affected in the presence of a co-ion. The uptake of copper by the biomass decreased in the presence of zinc and vice versa. The decrease in metal uptake was dependent on the concentrations of metals in the two-component aqueous solutions. The effect of copper on zinc uptake was more pronounced than the effect of zinc on copper uptake.

Mixed-ligand aroylhydrazone complexes of molybdenum: Synthesis, structure and biological activity

The reaction of the benzoylhydrazone of 2-hydroxybenzaldehyde (H2L) with MoO2(acac)(2)] proceeds smoothly in refluxing ethanol to afford an orange complex MoO2L(C2H5OH)] (1). The substrate binding capacity of 1 has been demonstrated by the formation and isolation of two mononuclear MoO2L(Q)] {where Q = imidazole (2a) and 1-methylimidazole (2b)} and one dinuclear (MoO2L)(2)(Q)] {Q = 4,4'-bipyridine (3)} mixed-ligand oxomolybdenum complex. All the complexes have been characterized by elemental analysis, magnetic and spectroscopic (IR, UV-Vis and NMR) measurements. The molecular structures of all the oxomolybdenum(VI) complexes (1, 2a, 2b and 3) have been determined by X-ray crystallography. In each complex, the dianionic planar ligand is coordinated to the metal centre via one enolate oxygen, one phenolate oxygen and an azomethine nitrogen atom. The complexes have been screened for their antibacterial activity against Escherichia coli, Bacillus and Pseudomonas aeruginosa. The minimum inhibitory concentration of these complexes and their antibacterial activity indicates that compounds 2a and 2b are potential lead molecules for drug designing. (C) 2012 Elsevier Ltd. All rights reserved.

Synthesis, structural studies and catalytic activity of dioxidomolybdenum(VI) complexes with aroylhydrazones of naphthol-derivative

Reaction of the salicylhydrazone of 2-hydroxy-1-naphthaldehyde (H2L1), anthranylhydrazone of 2hydroxy-l-naphthaldehyde (H2L2), benzoylhydrazone of 2-hydroxy-1-acetonaphthone (H2L3) and anthranylhydrazone of 2-hydroxy-1-acetonaphthone (H2L4; general abbreviation H2L) with MoO2(acac)21 afforded a series of 5- and 6- coordinate Mo(VI) complexes of the type MoO2L1-2(ROH)] where R = C2H5 (1) and CH3 (2)], and MoO2L3-4] (3 and 4). The substrate binding capacity of 1 has been demonstrated by the formation of one mononuclear mixed-ligand dioxidomolybdenum complex MoO2L1(Q)] (where Q= gamma-picoline (la)). Molecular structure of all the complexes (I, la, 2,3 and 4) is determined by X-ray crystallography, demonstrating the dibasic tridentate behavior of ligands. All the complexes show two irreversible reductive responses within the potential window -0.73 to -1.08 V, due to Movl/Mov and Mov/Mow processes. Catalytic potential of these complexes was tested for the oxidation of benzoin using 30% aqueous H2O2 as an oxidant in methanol. At least four reaction products, benzoic acid, benzaldehydedimethylacetal, methyl benzoate and benzil were obtained with the 95-99% conversion under optimized reaction conditions. Oxidative bromination of salicylaldehyde, a functional mimic of haloperoxidases, in aqueous 1-1202/KEr in the presence of HC1O4 at room temperature has also been carried out successfully. (C) 2013 Elsevier Ltd. All rights reserved.

Effect of substituent on the thermodynamics of D-glucopyranoside binding to concanavalin A, pea (Pisum sativum) lectin and lentil (Lens culinaris) lectin

Titration calorimetry measurements of the binding of phenyl-alpha (alpha PhOGlu), 3-methoxy (3MeOGlu), fluorodeoxy and deoxy derivatives of alpha-D-glucopyranose (Glu) to concanavalin A (conA), pea lectin and lentil lectin were performed at approx. 10 and 25 degrees C in 0.01 M dimethylglutaric acid/NaOH buffer, pH 6.9, containing 0.15 M NaCl and Mn2+ and Ca2+ ions. Apparently the 3-deoxy, 4-deoxy and 6-deoxy as well as the 4-fluorodeoxy and 6-fluorodeoxy derivatives of Glu do not bind to the lectins because no heat release was observed on the addition of aliquots of solutions of these derivatives to the lectin solutions. The binding enthalpies, delta H0b, and entropies, delta S0b, determined from the measurements were compared with the same thermodynamic binding parameters for Glu, D-mannopyranoside and methyl-alpha- D-glucopyranoside (alpha MeOGlu). The binding reactions are enthalpically driven with little change in the heat capacity on binding, and exhibit enthalpy-entropy compensation. Differences between the thermodynamic binding parameters can be rationalized in terms of the interactions apparent in the known crystal structures of the methyl-alpha-D-mannopyranoside-conA [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan and Campbell (1989) EMBO J. 8, 2189-2193] and pea lectin-trimanno-pyranoside [Rini, Hardman, Einspahr, Suddath and Carber (1993) J. Biol. Chem. 268, 10126-10132] complexes. Increases in the entropy change on binding are observed for alpha MeOGlu binding to pea and lentil lectin, for alpha PhOGlu binding to conA and pea lectin, and for 3MeOGlu binding to pea lectin relative to the entropy change for Glu binding, and imply that the phenoxy and methoxy substituents provide additional hydrophobic interactions in the complex. Increases in the binding enthalpy relative to that of Glu are observed for deoxy and fluoro derivatives in the C-1 and C-2 positions and imply that these substituents weaken the interaction with the surrounding water, thereby strengthening the interaction with the binding site.

Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.

Differential binding of peanut agglutinin with lipopolysaccharide of homologous and heterologous Rhizobium

To establish the crucial role of lipopolysaccharide in the initial recognition event of symbiotic peanut-Rhizobium system the ability of various surface polysaccharides isolated from Bradyrhizobium arachis to inhibit the precipitin reaction between peanut agglutinin and asialoganglioside: deoxycholate (1:1) micelles was estimated. It was compared with that of nonsymbiotic systems e.g. Bradyrhizobium japonicum, Bradyrhizobium ciceris and Escherichia coli. Peanut agglutinin was found to interact more strongly with the lipopolysaccharide of Bradyrhizobium arachis than the exopolysaccharide or capsular polysaccharide. The inhibitory capacity of lipopolysaccharides from homologous and heterologous Bradyrhizobium as measured in terms of the concentration necessary for 50 percent inhibition of precipitin reaction were 1428, 500, 410, and 277 times less than that of lactose for Bradyrhizobium arachis, B. japonicum, B. ciceris and Escherichia coli, respectively. These results support that host lectin peanut agglutinin can recognize homologous Bradyrhizobium lipopolysaccharide by virtue of its binding specificity of higher magnitude.

Reducibility of Ce1-xZrxO2: origin of enhanced oxygen storage capacity

We combine first-principles calculations with EXAFS studies to investigate the origin of high oxygen storage capacity in ceria-zirconia solid solution, prepared by solution combustion method. We find that nanocrystalline Ce0.5Zr0.5O2 can be reduced to Ce0.5Zr0.5O1.57 by H-2 upto 850 degrees C with an OSC of 65 cc/gm which is extremely high. Calculated local atomic-scale structure reveals the presence of long and short bonds resulting in four-fold coordination of the cations, confirmed by the EXAFS studies. Bond valence analysis of the microscopic structure and energetics is used to evaluate the strength of binding of different oxide ions and vacancies. We find the presence of strongly and weakly bound oxygens, of which the latter are responsible for the higher oxygen storage capacity in the mixed oxides than in the pure CeO2.

Thermodynamics of lectin-sugar interaction: Binding of sugars to winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBAI)

Combining site of WBAI is extended and encompasses all the residues of blood group A-reactive trisaccharide [GalNAcalpha3Galbeta4Glc]. Though both of the fucose residues of A-pentasaccharide [GalNAcalpha(Fucalpha2)3Galbeta(Fucalpha3)4Glc] do not directly interact, with the combining site they thermodynamically favour the interaction of GalNAcalpha3Galbeta4Glc part of the molecule by imposing a sterically favourable orientation of the binding epitope viz. GalNAcalpha3Galbeta4Glc of the saccharide. Binding of sugars is driven by enthalpy and is devoid of heat capacity changes. This together with enthalpy-entropy compensation observed for these processes underscore the importance of water reorganization as being one of the principal determinant of protein-sugar interactions.

Binding of DNA Nucleobases and Nucleosides with Graphene

Interaction of two different samples of graphene with DNA nucleobases and nucleosides is investigated by isothermal titration calorimetry. The relative interaction energies of the nucleobases decrease in the order guanine (G) > adenine (A) > cytosine (C) > thy mine (T) in aqueous solutions, although the positions of C and T seem to be interchangeable. The same trend is found with the nucleosides. Interaction energies of the A-T and G-C pairs are somewhere between those of the constituent bases. Theoretical calculations including van der Wools interaction and solvation energies give the trend G > A similar to T > C. The magnitudes of the interaction energies of the nucleobases with graphene are similar to those found with single-walled carbon nonotubes.

Solvent effect in molecular recognition: Determining binding constants in different solvents following an extraction based protocol

Semi-rigid molecular tweezers 1, 3 and 4 bind picric acid with more than tenfold increment in tetrachloromethane as compared to chloroform.

Thermodynamics of ligand (substrate end product) binding to endoxylanase from Chainia sp. (NCL-82-5-1): isothermal calorimetry and fluorescence titration studies

The binding of xylo-oligosaccharides to Chainia endoxylanase resulted in a decrease in fluorescence intensity of the enzyme with the formation of 1:1 complex. Equilibrium and thermodynamic parameters of ligand binding were determined by fluorescence titrations and titration calorimetry. The affinity of xylanase for the oligosaccharides increases in the order X-2 < X-3 < X-4 less than or equal to X-5. Contributions from the enthalpy towards the free energy change decreased with increasing chain length from X-2 to X-4, whereas an increase in entropy was observed, the change in enthalpy and entropy of binding being compensatory. The entropically driven binding process suggested that hydrophobic interactions as well as hydrogen bonds play a predominant role in ligand binding.

Difference spectroscopic studies on binding of Cibacron blue F3GA to ribosome inactivating proteins: effect of beta-mercaptoethanol on the interaction with ricin

The interaction of Cibacron blue F3GA with ribosome inactivating proteins, ricin, ricin A-chain and momordin has been investigated using difference absorption spectroscopy. Ricin was found to bind the dye with a 20- and 2-fold lower affinity than ricin A-chain and momordin, respectively. A time dependent increase in the amplitude of Cibacron blue difference spectrum in the presence of ricin was observed on addition of beta-mercaptoethanol. Analysis of the kinetic profile of this increase showed a biphasic phenomenon and the observed rates were found to be independent of the concentration of beta-mercaptoethanol. Kinetics of reduction of the intersubunit disulphide bond in ricin by beta-mercaptoethanol showed that reduction pet se is a second order reaction. Therefore, the observed changes in the difference spectra of Cibacron blue probably indicate a slow change in the conformation of ricin, triggered by reduction of the intersubunit disulphide bond.