Energetics of Intermolecular Hydrogen Bonds in a Hydrophobic Protein Cavity

This work explores the energetics of intermolecular H-bonds inside a hydrophobic protein cavity. Kinetic measurements were performed on the gaseous deprotonated ions (at the ?7 charge state) of complexes of bovine β-lactoglobulin (Lg) and three monohydroxylated analogs of palmitic acid (PA): 3-hydroxypalmitic acid (3-OHPA), 7-hydroxypalmitic acid (7-OHPA), and 16-hydroxypalmitic acid (16-OHPA). From the increase in the activation energy for the dissociation of the (Lg + X-OHPA)^7– ions, compared with that of the (Lg + PA)^7– ion, it is concluded that the –OH groups of the X-OHPA ligands participate in strong (5 – 11 kcal mol^–1) intermolecular H-bonds in the hydrophobic cavity of Lg. The results of molecular dynamics (MD) simulations suggest that the –OH groups of 3-OHPA and 16-OHPA act as H-bond donors and interact with backbone carbonyl oxygens, whereas the –OH group of 7-OHPA acts as both H-bond donor and acceptor with nearby side chains. The capacity for intermolecular H-bonds within the Lg cavity, as suggested by the gas-phase measurements, does not necessarily lead to enhanced binding in aqueous solution. The association constant (K_a) measured for 7-OHPA [(2.3 ± 0.2) × 10⁵ M^–1] is similar to the value for the PA [(3.8 ± 0.1) × 10⁵ M^–1]; K_a for 3-OHPA [(1.1 ± 0.3) × 10⁶ M^–1] is approximately three-times larger, whereas K_a for 16-OHPA [(2.3 ± 0.2) × 10⁴ M^–1] is an order of magnitude smaller. Taken together, the results of this study suggest that the energetic penalty to desolvating the ligand –OH groups, which is necessary for complex formation, is similar in magnitude to the energetic contribution of the intermolecular H-bonds.

Dissociation Quotients for Citric Acid in Aqueous Sodium Chloride Media to 150°C

The three molal dissociation quotients for citric acid were measured potentiometrically with a hydrogen-electrode concentration cell from 5 to 150°C in NaCl solutions at ionic strengths of 0.1, 0.3, 0.6, and 1 molal. The molal dissociation quotients and available literature data at infinite dilution were fitted by empirical equations in the all-anionic form involving an extended Debye-Hückel term and up to five adjustable parameters involving functions of temperature and ionic strength. This treatment yielded the following thermodynamic quantitites for the first dissociation equilibrium at 25°C: logK _1a=−3.127±0.002, ΔH _1a ^o =4.1±0.2 kJ-mol⁻¹, ΔS _1a ^o =−46.3±0.7 J-K⁻¹-mol⁻¹, and ΔCp _1a ^o =−162±7 J-K⁻¹-mol⁻¹; for the second acid dissociation equilibrium at 25°C: logK _2a =−4.759±0.001, ΔH _2a ^o =2.2±0.1, ΔS _2a ^o =−83.8±0.4, and ΔCp _2a ^o =−192±15, and for the third dissociation equilibrium at 25°C: logK _3a=−6.397±0.002, ΔH _3a ^o =−3.6±0.2, ΔS _3a ^o =−134.5±0.7, and ΔCp _3a ^o =−231±7.     

Evidence that water can reduce the kinetic stability of protein-hydrophobic ligand interactions

The first quantitative comparison of the thermal dissociation rate constants measured for protein-ligand complexes in their hydrated and dehydrated states is described. Rate constants, measured using surface plasmon resonance spectroscopy, are reported for the dissociation of the 1:1 complexes of bovine β-lactoglobulin (Lg) with the fatty acids (FA), palmitic acid (PA), and stearic acid (SA), in aqueous solution at pH 8 and at temperatures ranging from 5 to 45 °C. The rate constants are compared to values determined from time-resolved blackbody infrared radiative dissociation measurements for the gaseous deprotonated (Lg+FA)(n-) ions, where n = 6 and 7, at temperatures ranging from 25 to 66 °C. Notably, the hydrated (Lg+PA) complex is kinetically less stable than the corresponding gas phase (Lg+PA)(n-) ions at all temperatures investigated; the hydrated (Lg+SA) complex is kinetically less stable than the gaseous (Lg+SA)(n-) ions at temperatures <45 °C. The greater kinetic stability of the gaseous (Lg+FA)(n-) ions originates from significantly larger, by 11-12 kcal mol(-1), E(a) values. It is proposed that the differences in the dissociation E(a) values measured in solution and the gas phase reflect the differential hydration of the reactant and the dissociative transition state.     

Protein–Protein Binding Affinities in Solution Determined by Electrospray Mass Spectrometry

Electrospray ionization (ESI) allows the transfer of multi-protein complexes into the gas phase, thereby providing a simple approach for monitoring the stoichiometry of these noncovalent assemblies by mass spectrometry (MS). It remains unclear, however, whether the measured ion abundance ratios of free and bound species are suitable for determining solution-phase binding affinities (K _d values). Many types of mass spectrometers employ rf-only quadrupoles as ion guides. This work demonstrates that the settings used for these devices are a key factor for ensuring uniform transmission behavior, which is a prerequisite for meaningful affinity measurements. Using bovine β-lactoglobulin and hemoglobin as model systems, it is demonstrated that under carefully adjusted conditions the “direct” ESI-MS approach is capable of providing K _d values that are in good agreement with previously published solution-phase data. Of the several ion sources tested, a regular ESI emitter operated with pressure-driven flow at 1 μL min^–1 provided the most favorable results. Potential problems in these experiments include conformationally-induced differences in ionization efficiencies, inadvertent collision-induced dissociation, and ESI-induced clustering artifacts. A number of simple tests can be conducted to assess whether or not these factors are prevalent under the conditions used. In addition, the fidelity of the method can be scrutinized by performing measurements over a wide concentration range. Overall, this work supports the viability of the direct ESI-MS approach for determining binding affinities of protein–protein complexes in solution.     

Laws of complex formation in a series of azo-substituted pyrocatechol derivatives and their tin(II) complexes

Physicochemical properties of new reagents, azo-substituted pyrocatechol derivatives and their tin(II) complexes, are studied. The acid-base properties of the hydroxy groups (pK′_i, pK″_i), parameters of complex formation reactions (pH, temperature, time), and instability constants of the complexes formed (pK _i) are determined. Quantitative correlations between the dissociation constants (pK′_a) of the functional analytical group, and the electronic Hammett constant σ for a substituent (pK′_a-pH₅₀ of the complex formation reaction), as well as between pK′_a and instability constants of the complexes (pK _a), are established. The quantitative correlations established allow the prediction of the physicochemical properties of the reagents and tin(II) complexes with new reagents of this class with the same functional analytical group (FAG) but other substituents.     

The influence of cations on the dissociation behaviour of copolymers of propene and maleic acid and their modified products with taurine in salt-free solution

Potentiometric and conductometric titrations were used to study the dissociation behaviour of poly(propene-co-maleic acid) and poly(propene-co-maleic acid) modified with various contents of taurine (2-aminoethanesulfonic acid) in salt-free solution. Copolymers of propene and maleic acid with different molecular weights were titrated with LiOH, NaOH, and KOH. The influence of molecular weight on pK_a is ascertainable in both the first and second dissociation step. Of the various alkali metal cations studied, lithium had the most significant effect on the dissociation behaviour. The acidic strength decreased in the order lithium > sodium ≥ potassium. After insertion of sulfonic acid groups into poly(propene-co-maleic acid), the influence of strong acidic groups on the dissociation behaviour of carboxylic groups was studied. The contents of taurine were 10, 25, and 50 mol%. The second dissociation step was analyzed in this case. The pK_a values increased with increasing content of taurine for titrations with LiOH and KOH, but not NaOH. When NaOH was used, the pK_a decreases if the polymer was modified with 10% taurine. Higher taurine contents had no influence on the magnitude of pK_a. The results demonstrate the strong influence of short-range electrostatic interactions on the dissociation behaviour of weak polyacids. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 1949–1955, 1999     

(E)-2-Benzylidenebenzocyclanones, part VIII: spectrophotometric determination of pKa values of some natural and synthetic chalcones and their cyclic analogues

Abstract  

UV–Vis spectrophotometry was used to determine acid dissociation constant (pK _a) values of the natural flavonoids phloretin, phlorizin, naringenin, and naringin, as well as 4′-hydroxychalcone, 4′-(dimethylamino)chalcone, and their cyclic analogues. Comparison of the results with those previously reported for the natural flavonoids showed the applied method is a relatively straightforward and easy-to-perform technique for the determination of pK _a values of compounds with relatively low solubility. Comparative analysis of the pK _a values of the synthetic chalcones showed a strong correlation between the degree of conjugation and the acid strength of the respective compounds with different geometry. Our results provide further evidence that modification of the three-dimensional structure of open-chain bioactive compounds is the method of choice to modify not only their stereochemistry but also their physicochemical properties.     

Toward total structural analysis of cardiolipins: multiple-stage linear ion-trap mass spectrometry on the [M − 2H + 3Li]<Superscript>+</Superscript> ions

ESI multiple-stage linear ion-trap (LIT) mass spectrometric approaches for a near-complete structural characterization of cardiolipins (CLs), including identification of the fatty acyl substituents, assignment of the fatty acid substituents on the glycerol backbone, and location of the double-bond(s) or cyclopropyl group along the fatty acid chain are described. Upon collisionally activated dissociation (CAD) on the [M − 2H + 3Li]⁺ ions of CL in an ion-trap (MS²), two sets of fragment ions (designated as (a + 136) and (b + 136) ions) analogous to those previously reported for the [M − 2H + 3Na]⁺ ions were observed, leading to assignment of the phosphatidyl moieties attached to 1′- or 3′-position of the central glycerol. Further dissociation of the (a + 136) (or (b + 136)) ions (MS³) gives rise to the (a + 136 − R_{1(or 2)}CO₂Li) (or b + 136 − R_{1(or 2)}CO₂Li) ion pairs that identify the fatty acid moieties and their position on the glycerol backbone. This is followed by MS⁴ on the (a + 136 − R_{1(or 2)}CO₂Li) (or b + 136 − R_{1(or 2)}CO₂Li) ion to eliminate a tricylic glycerophosphate ester residue (136 Da) to yield the (a − R_{1(or 2)}CO₂Li) ion, which is then subjected to MS⁵. The MS5 spectrum contains the structural information that locates the double-bond(s) or cyclopropyl group of the fatty acid substituents. Finally, the subsequent MS⁶ on the dilithiated fatty acid ions generated from MS⁵ also yields feature ions that confirm the assignment.     

Second Dissociation Constant of H<Subscript>2</Subscript>Te and the Absorption Spectra of HTe<Superscript>–</Superscript>, Te<Superscript>2–</Superscript> and Te<Subscript>2</Subscript><Superscript>2–</Superscript> in Aqueous Solution

We have measured the second acid dissociation constant, K _2a , at several ionic strengths for hydrogen telluride (H₂Te) using the Charge Transfer to Solvent (CTTS) uv spectra of its anions HTe⁻ and Te²⁻. Since it is produced in our solutions, we have also determined the spectra of Te₂ ²⁻ both in the uv and in the visible regions. At 25 ^∘C, K _2a = (1.28 ± 0.02) × 10⁻¹² by extrapolation to zero ionic strength. Its value at an ionic strength equal to 0.5 mol.dm^-3 was estimated to be (8.7 ± 0.2) × 10⁻¹². The solution thermodynamics of these species are also discussed and comparisons are made to related acids.     

Carbohydrate–Lipid Interactions: Affinities of Methylmannose Polysaccharides for Lipids in Aqueous Solution

The interactions between 3‐O‐methyl‐mannose polysaccharides (MMPs), extracted from Mycobacterium smegmatis (consisting of a mixture of MMP‐10, ‐11, ‐12 and ‐13) or obtained by chemical synthesis (MMP‐5_s, ‐8_s, ‐11_s and ‐14_s), and linear saturated and unsaturated fatty acids (FAs), and a commercial mixture of naphthenic acids (NAs) in aqueous solution at 25 °C and pH 8.5 were quantified by electrospray ionization mass spectrometry (ESI‐MS). Association constants (K_a) for MMP binding to four FAs (myristic acid, palmitic acid, stearic acid and trans‐parinaric acid) were measured by using an indirect ESI‐MS assay, the “proxy protein” method. The K_a values are in the 10⁴–10⁵ M ^?1 range and, based on results obtained for the binding of the synthetic MMPs with palmitic acid, increase with the size of the carbohydrate. Notably, the measured affinity of the extracted MMPs for trans‐parinaric acid is two orders of magnitude smaller than the reported value, which was determined by using a fluorescence assay. Using a newly developed competitive binding assay, referred to as the “proxy protein/proxy ligand” ESI‐MS method, it was shown that MMPs bind specifically to NAs in aqueous solution, with apparent affinities of approximately (5×10⁴) M ^?1 for the mixture of NAs tested. This represents the first demonstration that MMPs can bind to hydrophobic species more complex than those containing linear alkyl/alkenyl chains. Moreover, the approach developed here represents a novel method for probing carbohydrate–lipid interactions.     

Sorbic Acid as an Alkylating Agent

A kinetic study of the alkylating potential of the sorbic acid + NaOH and sorbic acid + KOH systems was performed in 7:3 (volume/volume) water + dioxane solvent mixtures. The following conclusions were drawn. First, the sorbic acid + sorbate system shows alkylating activity on the nucleophile 4-(p-nitrobenzyl)pyridine (NBP), which is used as a trap for alkylating agents having nucleophilic characteristics similar to DNA bases. Second, the maximum alkylating capacity is observed in the pH = 5.0 to 6.5 range. Third, the alkylation reactions comply with the rate equation r=k _alk[H⁺][S][NBP]/(K _a +[H⁺]), with K _a being the dissociation constant of sorbic acid. Fourth, an enthalpy–entropy (ΔH ^#/ΔS ^#) compensation effect for activation quantities is observed by comparing NBP alkylation reactions due to sorbic acid + NaOH, sorbic acid + KOH, as well as potassium sorbate + HCl mixtures. Fifth, the results may help to establish suitable expiration times for products preserved with sorbic acid.     

Does in-source decay occur independent of the ionization process in matrix-assisted laser desorption?

The influence of the acidic and basic characters of constituent amino acid residues on the peptide fragment ions produced by in-source decay under matrix assisted laser desorption/ionization (MALDI) conditions has been studied using positive- and negative-ion experiments. Whereas the in-source decay spectra of peptides containing basic Arg and/or Lys residues near the N-terminus showed so-called c_n- and a_n-series ions in positive-ion mode, a peptide that has an acidic amino acid cluster near the N-terminus and a basic residue near the C-terminus characteristically formed y_n- and z_n-series ions in the positive-ion in-source decay spectrum. These results indicated that fragment ion series produced by in-source decay depend strongly upon the acidic and basic characters of the constituent amino acid residues and the near N- and C-termini. It was suggested that in-source decay processes occur intrinsically at NH–C^α and CO–NH bonds independent of the formation of molecular-related ions, and that the cleavages at the NH–C^α and CO–NH bonds occurred independently and were dependent on the matrix used.     

Regularities of complexation in the series of sorbents on the polystyrene matrix and their complexes with chromium(III) and manganese(II)

The physicochemical properties of polymeric complexing sorbents with the o,o′-dihydroxy-(1-azo-1′)-functional complexing group were studied. Optimal sorption parameters for chromium(III) and manganese(II) (medium acidity, temperature, time), the sorption capacity of the sorbents, and the stability constants (pK _a′)of the complexes were determined. Correlation relations were established between the dissociation constants (pK _a′) of the sorbent functional group and pH₅₀ of the complexation of the considered elements; between and the stability of the formed complexes (logβ); and between pK _a′ and the deprotonation energy (E _depr) of the sorbent hydroxy group located in the para-position to the introduced substituent. The established correlation relations are useful for targeted prediction of the physicochemical parameters of the sorbents, their complexes, and the sorption of manganese(II) and chromium(III) aimed at recovery and pre-concentration of these ions from objects with various chemical composition.     

Determination of aqueous acid‐dissociation constants of aspartic acid using PCM/DFT method

Determination of acid‐dissociation constants, pK_a, of aspartic acid in aqueous solution, using density functional theory calculations combined with the conductor‐like polarizable continuum model (CPCM) and with integral‐equation‐formalism polarizable continuum model (IEFPCM) based on the UAKS and UAHF radii, was carried out. The computed pK_a values derived from the CPCM and IEFPCM with UAKS cavity model of bare structures of the B3LYP/6‐31+G(d,p)‐optimized tetrahydrated structures of aspartic acid species are mostly close to the experimental pK_a values. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

Buffer Standards for the Physiological pH of the Zwitterionic Compound, ACES, from 5 to 55 °C

The values of the second dissociation constant, pK ₂, and related thermodynamic quantities of [N-(2-acetamido)-2-aminoethanesulfonic acid] (ACES) have already been reported over the temperature range 5 to 55 °C including 37 °C. This paper reports the pa _H values of four chloride ion free buffer solutions and eight buffer solutions with I=0.16 mol⋅kg⁻¹, matching closely that of the physiological sample. Conventional pa _H values for all twelve buffer solutions from 5 to 55 °C are reported. The residual liquid-junction potential correction for two widely used temperatures, 25 and 37 °C, has been made. The flowing-junction calomel cell method has been utilized to measure E _j , the liquid-junction potential. The operational pH values for four buffer solutions at 25 and 37 °C are calculated using the physiological phosphate buffer standard based on the NBS/NIST convention. These solutions are recommended as pH standards in the pH range of 6.8 to 7.2 for physiological fluids.     

Temperature dependency of thermal conductivity of solid phases for fatty acids

Thermal conductivity variations with temperature of solid phases for lauric acid (LA), myristic acid (MA), pivalic acid (PA), and stearic acid (SA) have been measured with radial heat-flow method. Temperature dependencies of the thermal conductivity for same organic materials have been obtained by linear regression analysis. From graphs of thermal conductivity versus temperature, the thermal conductivity of solid phase at their melting temperature and temperature coefficients of thermal conductivity for LA, MA, PA, and SA have been found to be 0.37, 0.39, 0.23, and 0.35 W K^?1 m^?1 and 0.00935, 0.00446, 0.01095, and 0.00295 K^?1, respectively. The ratios of thermal conductivity of liquid phase to thermal conductivity of solid phase for LA, MA, PA, and SA have also been measured to be 0.52, 0.48, 0.25, and 0.59, respectively, with a Bridgman-type directional solidification apparatus.     

Determination of thermodynamic pKa values of pharmaceuticals from five different groups using capillary electrophoresis

A determination of the thermodynamic acid dissociation constants (pK_a) of 22 frequently used pharmaceuticals using capillary electrophoresis in aqueous media is presented in this work. The investigated pharmaceuticals belong to different pharmacological groups: macrolides, fluoroquinolones, sulfonamides, β‐lactams, tetracyclines, and other miscellaneous pharmaceuticals. The electrophoretic mobilities of the investigated analytes were monitored in a pH range from 2.00 to 10.82. The data were fitted with an appropriate mathematical model using a nonlinear regression analysis to obtain pK_a values. Experimentally obtained data were well described by the mathematical model chosen for each analyte that was confirmed by r² values higher than 0.99 for most of the investigated analytes. Extrapolations to zero ionic strength were used to determine the thermodynamic pK_a values. Experimentally obtained acid dissociation constants were interpreted using structural formulae of investigated analytes and the moieties corresponding to specific pK_a were identified.     

Oil-Water Partitioning of a Synthetic Tetracarboxylic Acid as a Function of pH

The oil-water partitioning of a synthetic tetraacid acting as a model compound for indigenous C₈₀-C₈₂ ARN acids has been studied as a function of pH, ionic strength and type of monovalent counterion. Experimental data obtained with ultraviolet-visible and HPLC/UV analyses have been fitted to thermodynamic models based on one, two or four dissociation steps to obtain o/w partition coefficients (K _wo ) of the fully protonated acid between chloroform and aqueous solutions, and its apparent acidity constant(s), pK _a. As the study is conducted above the CMC of the tetraacid, in general high apparent acidity constants were obtained in the range from 6 to 8 resulting from micellization equilibria. K _wo values were obtained in the range from 10^?3 to 10^?4, and decreasing with increasing salinity. At 50 mM K⁺, no conclusions could be made regarding the number of distinguishable dissociation steps, while at higher ionic strength (184 mM and 452 mM K⁺) and at 184 mM Na⁺ a model with two dissociation steps provided good fits to the experimental data. The first step was found to be given by a pK _a ≈ 6.6–6.8 and the second dissociation step at pK _a values ≈ 7.8–8.3. The two-step mechanism supports previous results obtained by potentiometric titrations. No significant difference in the o/w behavior was observed when changing the counterion from potassium to sodium. The main partitioning of the tetraacid in the aqueous phase occurred above pH 8, where the fully deprotonated acid was formed.     

Release of nitric oxide from nitrated fatty acids: Insights from computational chemistry

Nitrated fatty acids (NO₂‐FAs) exhibit a variety of important biological attributes, including a nitric oxide (˙NO) donor and a cell‐signaling molecule. We investigated the mechanisms of fatty‐acid nitration, and the release of ˙NO from NO₂‐FAs. NO₂‐FAs are formed effectively by the addition of ˙NO₂, followed by either hydrogen abstraction or addition of a second NO₂. The latter reaction results in a vicinal nitronitrite ester form of FA, which isomerizes into vicinal nitrohydroxy FA via hydronium ion catalysis. The nitrohydroxy FAs exist in equilibria with NO₂‐FAs. Nitration of conjugated linoleic acid (cLA) was proved to be significantly more efficient than that of LA. In a nonaqueous environment, release of ˙NO from nitrite ester (ONO‐FA) was facilitated by ˙NO₂. Furthermore, the release of ˙NO from NO₂‐cLA is the most favorable in the nitrite ester mechanism. In an aqueous environment, the modified Nef reaction was shown to be feasible. In addition, the release of ˙NO from 10‐ and 12‐NO₂‐LA involves a larger reaction barrier and is more endergonic than those from 9‐ and 13‐NO₂‐LA.     

Quantum-chemical DFT study of acid properties of thiols RSH and ligand power of their anions RS<Superscript>−</Superscript>

The DFT calculations of electronic and geometry structure, dissociation energies of S-H and S-C bonds, and acid dissociation constants (pK _a) for ten RSH thiols were fulfilled. Calibration corrections to the calculated pK _a^DFT values, which allowed obtaining a good correspondence with experimental data were introduced. Donor power of thiolate anions RS- was estimated for the prediction of stability of their compounds with ions of d-elements.