Binding of 8-anilinonaphthalene sulfonate to dimeric and tetrameric concanavalin A: energetics and its implications on saccharide binding studied by isothermal titration calorimetry and spectroscopy

The binding of 8-anilinonaphthalene sulfonate to concanavalin A has been investigated. Isothermal titration calorimetry (ITC) and circular dichroism studies have been performed under different experimental conditions to understand the binding quantitatively and evaluate contributions of different forces responsible for it. Isothermal titration calorimetric results of concanavalin A with ANS at pH 5.2 and 2.5, where it exists as a dimer, indicated binding heterogeneity and two classes of noninteracting sites. Enhancement of the binding constants from native to pH 2.5 suggests that the ANS binding is strongly influenced by the protein charge and the favorable alteration in the structure of concanavalin A as suggested by near-UV CD results. No binding was observed with the tetrameric form of concanavalin A, indicating shielding of sites due to dimerization of canonical dimers. The results have also demonstrated existence of a hydrophobic binding site that is distinct from the saccharide binding site.     

Thermodynamic insights into the binding of ANS with the salt induced molten globule states of cytochrome c

The molten globule (MG) state or the A-state of cytochrome c (cyt c) has been induced by addition of salts sodium perchlorate (NaClO₄), sodium thiocyanate (NaSCN), and sodium sulphate (Na₂SO₄) at pH 2.0. Isothermal titration calorimetry (ITC) has been used for determining the energetics of binding of 8-anilino naphthalene sulphonate (ANS) to the salt induced A-state of cyt c, and the accompanying thermodynamic parameters have been analyzed to elucidate the nature of the interactions between ANS and the A-state of cyt c. Temperature dependent studies of the binding process reveal that the binding is not a two state process and there are more than a single type of interactions involved. Addition of a bulky salt tetraethylammonium bromide (TEAB) increases the stoichiometry of binding significantly, with a reduction in the binding affinity at a higher concentration. The results provide quantitative information on the binding of ANS to the salt induced molten globule states of cyt c. It is further inferred that the binding involves a combination of hydrophobic and electrostatic interactions.     

Photochemistry of the three carboxypyridines in water: a pH dependent reaction

The photochemistry of ortho, meta and para-carboxypyridines (pK(a)(1)= 1.0-2.1 and pK(a)(2)= 4.7-5.3) in aqueous medium was studied by laser-flash photolysis and product studies. At pH < pK(a)(1), hydroxylated compounds are produced with low quantum yields. Within the pH range 4-7, ortho and meta isomers undergo dimerization together with decarboxylation with a quantum yield showing a very sharp maximum around pK(a)(2)([small phi](max)= 0.09 and 0.01, respectively) while the para isomer is photostable. End-of-pulse transients assigned to triplet states were detected by laser-flash photolysis at pH < pK(a)(1) and pH > 4. Additionally, the carboxypyridinyl radicals were detected as secondary intermediates at pH < pK(a)(1) and 4 < pH < 7 and the OH-adduct radicals at pH < pK(a)(1). This is in favour of an electron transfer reaction between triplet and starting compound producing a charge transfer species. The radical anion would escape as carboxypyridinyl radical while the radical cation may add water at pH < pK(a)(1) yielding the OH-adduct radical or may undergo decarboxylation at pH > 4. The high quantum yield of phototransformation of the ortho isomer at pH > 4 is due to an easy decarboxylation process. A reaction scheme is proposed accounting for the dependences of [small phi] on both the pH and the carboxypyridines concentration. This study points out the distinct pattern of reactivity of carboxypyridines depending on the ionisation state of starting compounds and isomeric substitution.     

Effect of the pH value on the thermal stability of alkaline phosphatase

The thermal stability of bovine and chicken intestinal alkaline phosphatase (IAP) was studied at 60°C over a pH range from 6.7 to 9.0. It was shown that the peaks of stability (at pH 7.5) and activity (at pH 9.0) do not coincide. The pH dependence of the elementary rate constants of dissociative thermal inactivation of the IAP dimers was determined: k ₁ (the rate constant of dissociation of active dimer E₂) and k _d (the rate constant of denaturation of the inactive monomers). At pH 7.5, the stability IAP attains its highest level. As the pH increases, k ₁ increases drastically while k _d does so only slightly. A comparison of the rate constants of dissociation k ₁ and association k ₁ showed that the interrelation between these parameters explains why the stability of the active enzyme is lower at acidic and basic pH values. The pH produces a weaker effect on the stability of the inactive monomers. An analysis of the thermal stability of chicken IAP at an optimum pH value and 55–60°C showed that the thermal inactivation is a three-stage process (including dissociation and denaturation) with an induction period. Measuring the induction period makes it possible to determine the minimum number of latent stages preceding the dissociation of E₂: 6, 4, and 3 at 55, 58, and 60°C, respectively.     

Distribution of phenoxyacetic acid in two-phase organic solvent-water systems

The distribution of phenoxyacetic acid in two-phase organic solvent-water systems and its dimerization in the organic phase were studied. Organic solvents were normal aliphatic alcohols (C₅–C₁₀), benzene, and its derivatives. The distribution (K _dis) and dimerization (K _dim) constants of this acid were determined. The correlations of these quantities with physicochemical parameters of the organic solvents were revealed. The influence of the organic solvent polarity, pH of the aqueous phase, and acid concentration on the physicochemical behavior of the acid in the two-phase systems was analyzed.     

Fluorescence studies on the interaction of hydrophobic ligands with Momordica charantia (bitter gourd) seed lectin

The interaction of Momordica charantia (bitter gourd) seed lectin (MCL) with several nucleic acid bases has been investigated by monitoring changes induced in the protein fluorescence by ligand binding. Values of the binding constant, K_a were obtained as 1.1 × 10⁴, 1.56 × 10⁴ and 2.2 × 10³ M^?1 for adenine, cytosine and uracil, respectively. In addition, binding of 8-anilinonaphthalene 1-sulfonate (ANS) with MCL was investigated by fluorescence spectroscopy. Interaction with MCL at low pH results in a large enhancement of the fluorescence intensity of ANS with a concomitant blue shift in the emission λ_max, whereas at neutral and basic pH changes in both fluorescence intensity and emission maximum were very small, clearly suggesting that the MCL–ANS interaction is stronger at lower pH values. When excited at 295 nm in the presence of ANS, the protein fluorescence decreased with a concomitant increase in the emission intensity of ANS, suggesting resonance energy transfer from the tryptophan residues of MCL to ANS. Gel filtration profiles of MCL at pH values 2.0 and 7.4 are similar indicating that the tetrameric nature of MCL is retained even at low pH. Addition of lactose or adenine to MCL–ANS mixture did not alter the change in ANS fluorescence suggesting that lactose, adenine and ANS bind to MCL at independent and non-interacting sites. These results are relevant to understanding the functional role of MCL in the parent tissue.     

N-Hydroxyurea dimers: A matrix isolation and theoretical study

The dimerization of N-hydroxyurea (NH₂CONHOH) has been investigated by FTIR matrix isolation spectroscopy and DFT(B3LYP)/6-311++G(2d,2p) calculations. The analysis of NH₂CONHOH/Ar matrix spectra and comparison with theoretical ones reveal the formation of two types of dimers with a strong OH⋯O hydrogen bond. There is an additional weak interaction between the oxygen atom of the OH group of the proton donor molecule and the NH or NH₂ group of the proton acceptor in both dimers, respectively. The identified structures correspond to local minima on the PES. The formation of the less stable structures not the most stable ones indicates that the creation of N-hydroxyurea dimers is related to the dipole-dipole interaction at the initial stage of the dimerization process, which favours generation of polar dimers.     

Constantes d'acidite de l'acide pyrophosphorique et constantes de stabilite des complexes avec le potassium et le sodium: en|The acidity constants of pyrophosphoric acid and the stability constants of complexes with potassium and sodium ions

(The acidity constants of pyrophosphoric acid and the stability constants of complexes with potassium and sodium ions)The effect of potassium and sodium ions on the acidity constants of pyrophosphoric acid is studied by potentiometry at 25°C and at an ionic strength of 0.5 mol l^-1. Comparison of the results obtained in potassium chloride or sodium chloride medium with those found in tetramethylammonium chloride medium provides evidence of the formation of potassium and sodium complexes of the type MHP₂O^2-₇ and MP₂O^3-₇. Stability constants of these complexes are calculated and distribution diagrams of the species as a function of pH are given for the three different media utilized.     

Protolytic properties of some bis(dimethylaminomethyl)phenols in the presence of surfactants

Aggregation and protolytic properties of bis(dimethylaminomethyl)phenols containing methyl (HA) and nonyl (HL) substituents at the benzene ring are studied in aqueous solutions of isopropanol and various surfactants with potentiometric titration, tensiometry, and mathematical modeling of equilibria. Monomers, dimers, and tetramers of HA and HL are found. It is shown that the degree of compound aggregation depends on the solution concentration and pH. Sodium dodecyl sulfate and HA form associates, whereas SDS and HL form mixed micelles at the CMC-1 and CMC-2 critical micellization concentrations. In micellar solutions of Triton X-100 and cetyltrimethylammonium bromide, the mixed micelles are not found via tensiometry. Protonated species of tetramer, dimer, and monomer of investigated compounds are revealed, depending on the acidity of the medium. Phenolate forms of HA and HL do not exist under experimental conditions. Apparent protonation constants are determined and it is shown that, for the HA compound that does not form micelles, the protonation constants of the same-type species increase in the presence of the three surfactants used as compared to the water-isopropanol solution. Decreasing constants of analogous HL forms in the solutions of CTAB, nonionic surfactant (C _Tx = 10 mM), and SDS (pH > 7) are attributed to the formation of associates or mixed micelles of this compound and surfactants under experimental conditions.     

Double versus single helical structures of oligopyridine-dicarboxamide strands. Part 2: The role of side chains

A series of heptameric oligoamides comprising 4-alkoxy-substituted 2,6-diaminopyridine and 2,6-pyridine-dicarbonyl units have been synthesized using convergent methods. The hybridization of these compounds into double helical dimers was studied in solution by ¹H NMR spectroscopy in CDCl₃ or DMSO-d₆ at various concentrations, and in the solid state using X-ray crystallographic analysis. Both solid state and solution data suggest that these compounds follow identical hybridization schemes. In CDCl₃, the oligomers possess dimerization constants considerably (up to 2000-fold) higher than related compounds having no alkoxy substituents on their 2,6-diaminopyridine units. The origin of this effect can be in part interpreted as a result of interactions between the 4-alkoxy side chains when they are present on all pyridine rings. For example, 4-benzyloxy-substituted oligomer 2 has a higher dimerization constant than 4-decyloxy and 4-methoxy-substituted analogues 1 and 3. The crystal structure of 2 reveals multiple aromatic-aromatic interactions between the benzyl side chains, both face-to-face and edge-to-face at various angles surrounding the duplex. In the solid state, these double helices are stacked on top of each other to form long channels filled with water molecules. The 4-methoxy and 4-decyloxy-substituted analogues 1 and 3 have similar dimerization constants, showing that interactions between side chains are not significant between purely aliphatic residues. Consequently, the high stability of the double helices formed by 1 and 3 compared to related compounds having alkoxy substituents on their 2,6-pyridine-dicarbonyl units only does not find its origin in interactions between side chains but in the direct effect of the alkoxy substituents upon main chain aryl-aryl interactions.     

Non-radiative pathways of anilino-naphthalene sulphonates: Twisted intramolecular charge transfer versus intersystem crossing

The effect of solvent polarity and external heavy atom (ethyl iodide) on the fluorescence properties of 2-tolouidino-6-naphthalene-sulphonate (TNS) and 1-anilino-8-naphthalene-sulphonate (ANS) have been studied. For ANS with rise in polarity, a monotonic decrease of fluorescence yield (α_f) and lifetime (τ_f) is observed while ethyl iodide was found to have negligible effect. For TNS at low polarity α_f and τ_f are found to increase with rise in polarity, while ethyl iodide causes a decrease in τ_f. At high polarity [E_T(30) > 51] behaviour of TNS is more or less similar to that of ANS. It is proposed that polarity-dependent, twisted intramolecular charge transfer is the main non-radiative process for ANS at all polarities [E_T(30) = 36–63] and for TNS only at high polarity. At low polarity intersystem crossing is the main non-radiative pathway for TNS.     

Determination of the Dimerization Equilibrium Constants of Omeprazole and Pirkle's Alcohol through Optical‐Rotation Measurements

A new procedure to measure the equilibrium constants for the dimerization (homochiral and heterochiral) reactions of enantiomers in solution was applied to two different compounds, namely omeprazole and Pirkle's alcohol, both in CHCl₃. This procedure is based on the measurement of the optical rotation of the solution as a function of its composition, which exhibits a nonlinear dependence on the enantiomer enrichment. Such nonlinearity depends on the extent of dimerization, and it is a strong effect in the case of omeprazole, whose equilibrium constants are 14.0±3.5 l/mol and 25.2±4.0 l/mol for the formation of homochiral and of heterochiral dimers, respectively. Pirkle's alcohol exhibits a weaker effect that allows only to estimate the order of magnitude of these constants, i.e., ca. 0.10 and 0.34 l/mol, respectively.     

Synthesis of new di- and triamine diosgenin dimers

In this study, a high yield synthesis of symmetrical steroidal polyamine dimers was achieved by the dimerization of (25R)-3β-acetoxyfurost-5-en-26-al via several di- and triamine linkers under mild conditions. To ensure the dimerization via E-E ring, the hydroxyl group in diosgenin was protected by an acetyl group. The important step is opening the spiroketal moiety using NaCNBH₃/AcOH to furnish the primary alcohol at C-26, followed by oxidation using PCC/CH₂Cl₂ to synthesize the desired aldehyde. Finally, reductive amination with diaminopropane, diaminobutane, diaminohexane, and spermidine using Na(OAc)₃BH as reducing agent, afforded the required four dimers.     

Magnetic circular dichroism of molecules in dense media: Dimerization of benzoic acids

The purpose of this communication has been to show the application of MCD to dimerization of benzoic acid and its derivatives. We present results of the dimerization equilibrium constants from resolving the observed MCD spectra of the intramolecular charge transfer band (CT) into the spectra of monomers and dimers, and of the B-coefficients (or the magnetic rotational strengths) of the CT band of MCD of monomers and dimers.     

Potentiometric Studies on the Equilibria of Flufenamic Acid in Aqueous Solutions and in Two-phase Organic Solvent + Water Systems

In this paper, the flufenamic acid equilibria in aqueous solution and in two-phase organic solvent + aqueous solution are described and presented. The dissociation constants K _a1 and K _a2 were determined in MDM + water mixtures. The Yashuda-Shedlovsky extrapolation procedure has been used to obtain the values of K _a1 and K _a2 in aqueous solutions. The distribution ratio D was measured in the toluene + water system over a wide range of pH by the shaking flask method. Based on the results of potentiometric titrations in two-phase organic solvent (benzene, ethylbenzene, toluene, carbon tetrachloride, chloroform, chlorobenzene, and bromobenzene) + aqueous systems, and using models of single and multistep equilibria, the values of distribution constants K _D and dimerization constants K _dim were calculated. The influences of polarity of the applied solvents and pH of the aqueous phase, on the speciation of the particular forms of flufenamic acid in both phases, were demonstrated.     

Inhibitory effect of different product analogues on β-alanine synthase: A thermodynamic and fluorescence analysis

The enzyme N-carbamoyl-β-alanine amidohydrolase catalyse the hydrolysis of N-carbamoyl-β-alanine or N-carbamoyl-β-aminoisobutyric acid to β-alanine or 3-aminoisobutyric acid, under the release of carbon-dioxide and ammonia. This work studies the inhibition of N-carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens C58 (Atβcar) by different carboxylic acid compounds that differ in number of carbons, and position and size of ramification, while the binding thermodynamics of the inhibitors are studied by isothermal titration calorimetry (ITC) and fluorescence. From the binding constants and inhibition studies, we conclude that propionate is the most efficient inhibitor among those tested. Substitution of the linear alkyl acids in positions 2 and 3 resulted in a drastic decrease of the affinity. The thermodynamic parameters show that a conformational change is triggered upon ligand binding. Binding enthalpy ΔH_b is negative in all cases for all ligands, and thus, Van der Waals interactions and hydrogen bonding are most probably the major sources for this term. The process is entropically favoured at all temperatures and pH studied, most probably due to the liberation of water molecules accompanying the conformational change of the enzyme.     

The oxidation of 2-alkoxy-3,6-di-tert-butylphenols. The reversible dimerization of 2-alkoxy-3,6-di-tert-butylphenoxy radicals

The oxidation of 2-alkoxy-3,6-di-tert-butylphenols has been studied. It was found that 2-RO-3,6-di-tert-butylphenoxy radicals (R = Et, Pr, Ph) undergo dimerization by C-O coupling. The X-ray structure of 2-ethoxy-3,6-di-tert-butylphenoxy dimer has been determined. Radical dimers dissociate reversibly in solution to give two phenoxy radicals at 200-350 K. By using EPR spectroscopy the equilibrium concentration of the phenoxy radicals, equilibrium constants and ΔH and ΔS of dissociation have been determined.     

Complex formation of gallium(III) with 8-hydroxy-7-iodoquinoline-5-sulphonic acid (ferron)

The complex formation of gallium(III) with 8-hydroxy-7-iodoquinoline-5-sulphonic acid (ferron) (L) was studied by potentiometry; and the dissociation constants of the ligand were redetermined. A new computer program made it possible to make allowance for a considerable number of equilibria. In the pH range 2–10, the main species were as follows (the logarithm of the absolute stability constants at 25.0 ±0.1° for ionic strength 0.1 M are given in parentheses): GaHL (11.3), GaL (14.7), Ga(OH)₂L (32.3), GaL₂ (23.9), GaOHL₂ (31.0) and GaL₃ (29.6). For 1:1, 1:2 and 1:3 mixtures of metal with ferron, the mole fractions of the various complexes as a function of pH were calculated; the effect of pH on the conditional constants of the species GaL, GaL₂ and GaL₃ was also established.     

Kinetic and mechanistic study of the reduction of 2,6-dichlorophenol indophenol by dithionites

The reaction of 2,6-dichlorophenolindophenol (DCPI) and dithionites is studied kinetically by applying the stopped-flow technique. Reaction rate constants are given for the pH range 1.30–6.80. The reaction was found to follow first-order kinetics with respect to each of the reactants. For pH 3.97, 5.10 and 6.80, the second-order reaction rate constant was determined by applying four different technique. Mean values of k = 172±5, 200±2 and 276±4 l mol^?1s^?1 are given for pH 3.97, 5.10 and 6.80, respectively. A mechanism is proposed for the reaction, which suggests partial reactions of all possible species of DCPI and dithionites at any pH. An equation for the calculation of k at any pH is derived, which gives k as a function of [H⁺], the partial reaction rate constants and the dissociation constants of DCPI and H₂S₂O₄. Values of reaction rate constants of all possible partial reactions are also presented.     

Synthesis of 13c-labelled [s]-proline and its conformational analysis by nuclear magnetic resonance

In order to analyze completely the ring conformation of [S]-Proline (l-thiazolidine-4-carboxylic acid) this compound has been synthesized with its C_δ atom enriched at 90% in ¹³C. The ¹H-¹H, ¹³C-¹H, ¹³C-¹³C vicinal coupling constants measured at several pH values describe well the geometry of the thiazolidine ring. The results suggest that the ring has an average planar conformation only in a narrow range of pH close to the pK of the amine group. Above and below this pH the ring tends to adopt preferentially the S_γendo and S_γexo puckered conformations respectively. It is concluded that a good correlation exists between the value of the dihedral angle ø and the character S_γendo or S_γexo of the ring.