Revisiting the hydrolysis of ampicillin catalyzed by Temoneira-1 ß-lactamase, and the effect of Ni(II), Cd(II) and Hg(II).

ß-Lactamases grant resistance to bacteria against ß-lactam antibiotics. The active center of TEM-1 ß-lactamase accommodates a Ser-Xaa-Xaa-Lys motif. TEM-1 ß-lactamase is not a metalloenzyme but it possesses several putative metal ion binding sites. The sites composed of His residue pairs chelate borderline transition metal ions such as Ni(II). In addition, there are many sulfur-containing donor groups that can coordinate soft metal ions such as Hg(II). Cd(II) may bind to both types of the above listed donor groups. No significant change was observed in the circular dichroism spectra of TEM-1 ß-lactamase on increasing the metal ion content of the samples, with the exception of Hg(II) inducing a small change in the secondary structure of the protein. A weak nonspecific binding of Hg(II) was proven by mass spectrometry and 119m Hg perturbed angular correlation spectroscopy. The hydrolytic process of ampicillin catalyzed by TEM-1 ß-lactamase was described by the kinetic analysis of the set of full catalytic progress curves, where the slow, yet observable conversion of the primary reaction product into a second one, identified as ampilloic acid by mass spectrometry, needed also to be considered in the applied model. Ni(II) and Cd(II) slightly promoted the catalytic activity of the enzyme while Hg(II) exerted a noticeable inhibitory effect. Hg(II) and Ni(II), applied at 10 µM concentration, inhibited the growth of E. coli BL21(DE3) in M9 minimal medium in the absence of ampicillin, but addition of the antibiotic could neutralize this toxic effect by complexing the metal ions.

Equilibria and Dynamics of Sodium Citrate Aqueous Solutions: The Hydration of Citrate and Formation of the Na3Cit0 Ion Aggregate.

Sodium citrate (Na3Cit) has a crucial role in many biological and industrial processes. Yet, quantitative information on its hydration and the ion association between Na+ and Cit3- ions in a broad range of salt concentrations is still lacking. In this work, we study both ion association equilibria and relaxation dynamics of sodium citrate solutions by combining potentiometry, spectrophotometry, and dielectric spectroscopy. From photometric and potentiometric measurements, we detect the formation of the NaCit2- ion-pair and the neutral Na3Cit0 ion aggregate in a wide range of ionic strengths (0.5-4 M). Due to its remarkable stability, the latter becomes the prevailing species at higher salt concentrations. In the dielectric spectra, we observe the dipolar relaxation of Cit3- and NaCit2- and two solvent-related processes, associated with the collective rearrangement of the H-bond network (cooperative water mode) and the H-bond flip of water molecules (fast water mode). Unlike numerous other salt solutions, the relaxation time of the cooperative mode scales with the viscosity indicating that the strongly hydrated anion fits well into the water network. That is, the stabilizing effect of anion-solvent interactions on the H-bond network outweighs the destructive impact of the cations as the latter are only present at low concentration, due to strong ion association. In conclusion, the affinity of citrate toward Na+ binding not only governs solution equilibria but also has a strong impact on water dynamics.

Magnesium(II) d-Gluconate Complexes Relevant to Radioactive Waste Disposals: Metal-Ion-Induced Ligand Deprotonation or Ligand-Promoted Metal-Ion Hydrolysis?

The complexation equilibria between Mg2+ and d-gluconate (Gluc-) ions are of particular importance in modeling the chemical speciation in low- and intermediate-level radioactive waste repositories. NMR measurements and potentiometric titrations conducted at 25 °C and 4 M ionic strength revealed the formation of the MgGluc+, MgGlucOH0, MgGluc(OH)2-, and Mg3Gluc2(OH)40 complexes. The trinuclear species provides indirect evidence for the existence of multinuclear magnesium(II) hydroxido complexes, whose formation was proposed earlier but has not been confirmed yet. Additionally, speciation calculations demonstrated that MgCl2 can markedly decrease the solubility of thorium(IV) at low ligand concentrations. Regarding the structure of MgGluc+, both IR spectra and density functional theory (DFT) calculations indicate the monodentate coordination of Gluc-. By the potentiometric data, the acidity of the water molecules is higher in the MgGluc+ and MgGlucOH0 species than in the Mg(H2O)62+ aqua ion. On the basis of DFT calculations, this ligand-promoted hydrolysis is caused by strong hydrogen bonds forming between Gluc- and Mg(H2O)62+. Conversely, metal-ion-induced ligand deprotonation takes place in the case of calcium(II) complexes, giving rise to salient variations on the NMR spectra in a strongly alkaline medium.

The acidity and self-catalyzed lactonization of l-gulonic acid: Thermodynamic, kinetic and computational study.

Lactonization and proton dissociation of sugar acids take place simultaneously in acidic aqueous solutions. The protonation-deprotonation processes are always fast, whilst the formation and hydrolysis of γ- and δ-lactones are usually slower. Thus, both thermodynamic and kinetic information are required for the complete understanding of these reactions. The protonation constant (Kp) of l-gulonate (Gul-) was determined from potentiometric and polarimetric measurements, while the individual lactonization constants (KL,γ and KL,δ) for l-gulonic acid (HGul) were obtained via13C NMR experiments. The applicability of this method was proven by measuring these well-known constants for d-gluconic acid (HGluc) and by comparing them to literature data. l-gulonic acid γ-lactone (γ-HGul) has remarkable stability in contrast with δ-HGul as well as γ- and δ-HGluc. The polarimetric measurement implies that the main factor responsible for the enhanced stability of γ-HGul is that its hydrolysis is much slower than that of δ-HGul. This higher stability of the γ-HGul ring over its δ-isomer was also confirmed by quantum chemical calculations. A new confirmed feature of the reaction is that in parallel to H3O+, HGul also catalyzes the formation and reverse hydrolytic processes of γ-HGul, similarly to other general acid catalysts.

Configuration-dependent complex formation between Ca(II) and sugar carboxylate ligands in alkaline medium: Comparison of L-gulonate with D-gluconate and D-heptaguconate.

The calcium sugar carboxylate interactions in hyperalkaline solutions are of relevance in radioactive waste repositories and in certain industrial processes. The complex formation between L-gulonate and Ca2+ ions was studied in strongly alkaline medium at 25 °C and 1 M ionic strength and was compared with previous results reported for D-gluconate and D-heptagluconate. The deprotonation of the ligand was confirmed by potentiometric and 13C NMR spectroscopic measurements. Pronounced pH effects were seen in the presence of Ca2+ indicating strong complex formation. By the evaluation of the experimental data, two highly stable trinuclear species, Ca3Gul2H-3+ and the Ca3Gul2H-40, are formed in alkaline aqueous solutions. Polarimetric as well as 1H NMR spectroscopic measurements attested that the increased complex stability was due to the formation of strong metal ion - alcoholate interactions. Moreover, the 1H NMR spectra of the three anions refer to the role of configuration in metal ion-binding. That is, the participation of the C3-OH or C4-OH group is governed by the relative position (i.e., threo or erythro) of the C2-OH and C3-OH groups.

Clarifying the Equilibrium Speciation of Periodate Ions in Aqueous Medium.

Equilibria of periodate ion were reinvestigated in aqueous solution by using potentiometric titration, UV and Raman spectroscopies, and gravimetry simultaneously at 0.5 M ionic strength and at 25.0 ± 0.2 °C. Stepwise acid dissociation constants of orthoperiodic acid were found to be pK1 = 0.98 ± 0.18, pK2 = 7.42 ± 0.03, and pK3 = 10.99 ± 0.02, as well as pK2 = 7.55 ± 0.04 and pK3 = 11.25 ± 0.03 in the presence of sodium nitrate and sodium perchlorate as background salts, respectively. pK1 cannot be determined unambiguously from our experiments in the presence of sodium perchlorate. The molar absorptivity spectrum of H4IO6- and H3IO62- was determined in the range of 215-335 nm, as major species of periodate present from slightly acidic to slightly alkaline conditions. The solubility of periodate decreases significantly under alkaline conditions, and it was determined to be (2.8 ± 0.4) mM by gravimetry, under our experimental conditions. None of these studies gave any clear evidence for an ortho-meta equilibrium and the frequently invoked dimerization of periodate. All measurements can quantitatively be described by the presence of orthoperiodic acid and its three successive deprotonation steps.

Exploring the boundaries of direct detection and characterization of labile isomers - a case study of copper(ii)-dipeptide systems.

The investigation of the linkage isomers of biologically essential and kinetically labile metal complexes in aqueous solutions poses a challenge, as these microspecies cannot be separately studied. Therefore, derivatives are commonly used to initially determine the stability or spectral characteristics of at least one of the isomers. Here we directly detect the isomers, describe the metal ion coordination sphere, speciation and thermodynamic parameters by a synergistic application of temperature dependent EPR and CD spectroscopic measurements in copper(ii)-dipeptide systems including His-Gly and His-Ala ligands. The ΔH = (-23 ± 4) kJ mol-1 value of the standard enthalpy change corresponding to the peptide-type to histamine-type isomerisation equilibrium of the [CuL]+ complex was corroborated by several techniques. The preferential coordination of the side-chains was observed at lower temperatures, whereas, metal-binding of the backbone atoms became favourable upon increasing temperature. This study exemplifies the necessity of using temperature dependent multiple methodologies for a reliable description of similar systems for upstream applications.

Formation of mono- and binuclear neodymium(iii)-gluconate complexes in aqueous solutions in the pH range of 2-8.

The complex formation between Nd(iii) and d-gluconate (Gluc-) is of relevance in modelling the chemical equilibria of radioactive waste repositories. In the present work, the formation of NdpGlucqH-r complexes at 25 °C and pH = 2-8 was studied via spectrophotometry, potentiometry, freezing point depression, conductometry and NMR spectroscopy. In addition to the four mononuclear complexes (pq-r = 110, 120, 130 and 11-2), the formation of two binuclear, so far unknown complexes (pq-r = 23-2 and 24-2) was revealed. Between pH = 5.5 and 7, with the increasing metal ion and ligand concentrations, the Nd2Gluc3H-2+ species becomes progressively predominant. Under the conditions characteristic of waste repositories, however, the formation of these complexes can be neglected. Regarding the binding sites of Gluc-, C2-OH and C3-OH groups, in addition to the carboxylate ion, were identified from 1H and 13C spectroscopic measurements. Above pH = 6, the metal-ligand interactions became stronger implying the formation of deprotonated complexes involving the C2-OH group, while the displacement of the second proton at the C3-OH is also possible. The metal ion induced deprotonation of the ligand was confirmed by DFT calculations.

Calcium complexation and acid-base properties of l-gulonate, a diastereomer of d-gluconate.

The Ca(ii)-complexation and acid-base properties of l-gulonic acid (HGul), a diastereomer of d-gluconic acid (HGluc) differing only in the configurations of C2 and C5 have been investigated via1H and 13C NMR spectroscopies, Ca-ISE- and pH-potentiometry, polarimetry and freezing point depression. Data obtained for Gul-/HGul have been compared with those of Gluc-/HGluc. It was found that some properties (acid dissociation constant, the stoichiometry and formation constants of the Ca(ii)-complexes) were insensitive to the difference in the configuration. In solutions with pH close to neutral, the presence of the complexes CaGul+ and CaGul20 was unambiguously proven, with formation constants of log K1,1 = 0.88 ± 0.02 and log ß1,2 = 1.51 ± 0.03 (I = 1 M, T = 25 °C). The formation of Ca(Gluc)20 was also observed by others, which implies that the formation of the charge neutral 1 : 2 Ca(ii)-complex of sugar carboxylates is more common than was previously believed. The stability of these species was found not to vary significantly in the ionic strength range of 1-4 M. Polarimetric measurements attested that the structure of Gul- did not change markedly upon complexation. NMR experiments suggest the coordination of C2-OH and C3-OH groups (beside COO-). DFT calculations support the existence of two coordination isomers, in which Ca2+ is attached to the COO-, C2-OH and C3-OH (in agreement with NMR), as well as to the COO-, C3-OH and C4-OH groups.

Calcium l-tartrate complex formation in neutral and in hyperalkaline aqueous solutions.

The complex formation reaction between the l-tartrate (Tar2-) and calcium ions taking place in neutral and in hyperalkaline (pH > 13) aqueous solutions has been investigated. It was demonstrated that upon NaOH addition the solubility of the CaTar(s) precipitate significantly increases. Conductometric and freezing point depression measurements further confirmed that in this process water soluble species are formed as a result of a reaction between the CaTar(s) and the hydroxide ion (or, conversely, between Ca(OH)2(s) and the Tar2- ion). 13C NMR spectroscopic measurements yielded the value of pK3 = 15.4 ± 0.2 for the proton dissociation of one of the alcoholic OH groups of Tar2- (at 25.0 °C and 4 M Na(Cl) ionic strength). Upon addition of calcium ions to an alkaline Tar2- solution, the 1H NMR signal gradually broadened and the 13C-satellite peaks split to two components, which also indicate complexation. From H2/Pt potentiometric titrations performed with solutions in the 13.6 ≤ pH ≤ 14.4 range, it was observed, that this complex formation is accompanied by a hydroxide ion consuming process. The titration curves can be best described via assuming the formation of the CaTarH-1-(aq) (lg ß11-1 = -11.2 ± 0.1) and CaTarH-22-(aq) (lg ß11-2 = -25.3 ± 0.1) complexes. In hyperalkaline solutions, these two species account for more than 90-99% of the calcium ions present and the contribution of the other reasonable and well-established calcium-containing solution species is rather small. The possible structures of the above complexes have been modeled via ab initio calculations. The stoichiometries are consistent both with species containing coordinated alcoholate group(s) and with mixed Ca(ii)-hydroxo-tartrato complexes. From the data available at present, both types of structures can be considered as chemically reasonable.

Speciation and structure of tin(II) in hyper-alkaline aqueous solution.

The identity of the predominating tin(ii)-hydroxide complex formed in hyper-alkaline aqueous solutions (0.2 ≤CNaOH≤ 12 mol dm(-3)) is determined by potentiometric titrations, Raman, Mössbauer and XANES spectroscopy, supplemented by quantum chemical calculations. Thermodynamic studies using a H2/Pt electrode up to free hydroxide concentrations of 1 mol dm(-3) showed the presence of a single monomeric complex with a tin(II) : hydroxide ratio of 1 : 3. This observation together with Raman and Mössbauer spectroscopic measurements supplemented by quantum mechanical calculations proved that the predominating complex is [Sn(OH)3](-), and that the presence of the other possible complex, [SnO(OH)](-), could not be proven with either experiments or simulations. The structure of the trihydroxidostannate(II) complex, [Sn(OH)3](-), was determined by EXAFS and was found to be independent of the applied hydroxide and tin(II) concentrations. The mean Sn-O bond distance is short, 2.078 Å, and in very good agreement with the only structure reported in the solid state. It is also shown that at pH values above 13 the speciation of the predominant trihydroxidostannate(II) complex is not affected by the presence of high concentrations of chloride ions.

Multinuclear complex formation between Ca(II) and gluconate ions in hyperalkaline solutions.

Alkaline solutions containing polyhydroxy carboxylates and Ca(II) are typical in cementitious radioactive waste repositories. Gluconate (Gluc(-)) is a structural and functional representative of these sugar carboxylates. In the current study, the structure and equilibria of complexes forming in such strongly alkaline solutions containing Ca(2+) and gluconate have been studied. It was found that Gluc(-) significantly increases the solubility of portlandite (Ca(OH)2(s)) under these conditions and Ca(2+) complexes of unexpectedly high stability are formed. The mononuclear (CaGluc(+) and [CaGlucOH](0)) complexes were found to be minor species, and predominant multinuclear complexes were identified. The formation of the neutral [Ca2Gluc(OH)3](0) (log ß213 = 8.03) and [Ca3Gluc2(OH)4](0) (log ß324 = 12.39) has been proven via H2/Pt-electrode potentiometric measurements and was confirmed via XAS, (1)H NMR, ESI-MS, conductometry, and freezing-point depression experiments. The binding sites of Gluc(-) were identified from multinuclear NMR measurements. Besides the carboxylate group, the O atoms on the second and third carbon atoms were proved to be the most probable sites for Ca(2+) binding. The suggested structure of the trinuclear complex was deduced from ab initio calculations. These observations are of relevance in the thermodynamic modeling of radioactive waste repositories, where the predominance of the binuclear Ca(2+) complex, which is a precursor of various high-stability ternary complexes with actinides, is demonstrated.

Using low-frequency IR spectra for the unambiguous identification of metal ion-ligand coordination sites in purpose-built complexes.

One of the aims of our long-term research is the identification of metal ion-ligand coordination sites in bioinspired metal ion-C- or N-protected amino acid (histidine, tyrosine, cysteine or cystine) complexes immobilised on the surface of chloropropylated silica gel or Merrifield resin. In an attempt to reach this goal, structurally related, but much simpler complexes have been prepared and their metal ion-ligand vibrations were determined from their low-frequency IR spectra. The central ions were Mn(II), Co(II), Ni(II) or Cu(II) and the ligands (imidazole, isopropylamine, monosodium malonate) were chosen to possess only one-type of potential donor group. The low-frequency IR spectra were taken of the complexes for each ion-ligand combination and the typical metal ion-functional group vibration bands were selected and identified. The usefulness of the obtained assignments is demonstrated on exemplary immobilised metal ion-protected amino acid complexes.

Complexation of Al(III) with gluconate in alkaline to hyperalkaline solutions: formation, stability and structure.

Contrary to suggestions in the literature, it has been proven that Al(III) forms a 1 : 1 complex with gluconate (hereafter Gluc(-)) in strongly alkaline (pH > 12) aqueous solutions. The complex formation was proven via(27)Al and (1)H NMR, freezing-point depression, polarimetric measurements as well as potentiometric and conductometric titrations. This complexation is a pH independent process, i.e., a condensation reaction takes place. The stability constant of the complex formed was derived from (1)H NMR and polarimetric measurements, and was found to be log K = 2.4 ± 0.4. In the complex formed, Al(III) has a tetrahedral geometry, and the Al(OH)4(-) is most probably statistically distributed between the alcoholate groups of the Gluc(-).

Multinuclear complex formation in aqueous solutions of Ca(II) and heptagluconate ions.

The equilibria and structure of complexes formed between the Ca(2+) ion and the heptagluconate (Hglu(-)) ion in both neutral and alkaline solutions have been studied. In alkaline solutions an uncharged, multinuclear complex is formed with the composition of Ca3Hglu2(OH)4 (or [Ca3Hglu2H(-4)](0)) with an unexpectedly high stability constant (lg ß(32-4) = 14.09). The formation of the trinuclear complex was deduced from potentiometry and confirmed by freezing-point depression measurements and conductometry as well. The binding sites of Hglu(-) were determined from NMR measurements. Besides the carboxylate group, the O atoms on the second and third carbon atoms proved to be the most probable sites for Ca(2+) binding.

An improved chemical model for the quantitative description of the front propagation in the tetrathionate-chlorite reaction.

It is experimentally proven that the stoichiometry of the tetrathionate-chlorite reaction is 2S4O(2-)6 + 8(1/2)ClO-(2) + 6H2O = 8SO(2-)4 + ClO-(3) + 7(1/2)Cl- + 12H+ near 1:4 molar ratio of the reactants. Re-evaluation of the previously measured front velocity--concentration curves also shows that this stoichiometry along with both the rate equation r = (1.6 x 10(5) M(-3) s(-1) [H+]2 + 3.6 x 10(7) M(-4) s(-1) [H+]3)[S4O(2-)6][ClO-(2)] and the protonation processes existing in the present system allow us to describe the front velocity as a function of the initial concentration of the reactants quantitatively. Some consequences detailed in the conclusions may concern not only uniquely the tetrathionate-chlorite reaction but any front propagation study including H+ as an autocatalyst.

Effect of chloride ion on the kinetics and mechanism of the reaction between chlorite ion and hypochlorous acid.

The effect of chloride ion on the chlorine dioxide formation in the ClO 2 (-)-HOCl reaction was studied by following .ClO 2 concentration spectrophotometrically at pH 5-6 in 0.5 M sodium acetate. On the basis of the earlier experimental data collected without initially added chloride and on new experiments, the earlier kinetic model was modified and extended to interpret the two series of experiments together. It was found that the chloride ion significantly increases the initial rate of .ClO 2 formation. At the same time, the .ClO 2 yield is increased in HOCl but decreased in ClO 2 (-) excess by the increase of the chloride ion concentration. The two-step hydrolysis of dissolved chlorine through Cl 2 + H 2O left harpoon over right harpoon Cl 2OH (-) + H (+) and Cl 2OH (-) left harpoon over right harpoon HOCl + Cl (-) and the increased reactivity of Cl 2OH (-) compared to HOCl are proposed to explain these phenomena. It is reinforced that the hydrolysis of the transient Cl 2O 2 takes place through a HOCl-catalyzed step instead of the spontaneous hydrolysis. A seven-step kinetic model with six rate parameters (constants and/or ratio of constants) is proposed on the basis of the rigorous least-squares fitting of the parameters simultaneously to 129 absorbance versus time curves measured up to approximately 90% conversion. The advantage of this method of evaluation is briefly outlined.

Inherent pitfalls in the simplified evaluation of kinetic curves.

It is shown and explained in detail by four examples generated from known kinetic models that simplified evaluation procedures--initial rate studies, individual exponential curve fitting method--may inherently lead to inappropriate chemical conclusions, even in the case of relatively simple kinetic systems. It is also shown that in the case of all four examples the simultaneous curve fitting immediately reveals the defectiveness of the kinetic model obtained from the simplified evaluation procedures. We therefore propose the extensive usage of the simultaneous curve fitting of all the kinetic traces to avoid these pitfalls and to find the appropriate kinetic models.

Chemical speciation in concentrated alkaline aluminate solutions in sodium, potassium and caesium media. Interpretation of the unusual variations of the observed hydroxide activity.

A detailed electrochemical investigation using H2/Pt electrode potentiometry as well as Raman and NMR spectroscopy was carried out to develop a comprehensive chemical explanation for the unusual patterns of hydroxide concentrations observed in strongly alkaline, highly concentrated aluminate solutions (Bayer-liquors). For this, aluminate solutions with various alkaline metal background cations were investigated. The effect of the temperature on the observed patterns was also studied, and for comparison with solutions of similar concentrations, the chemical speciation of borate solutions was also studied. The formation of the NaOH 0 ion-pair has been proven with the formation constant (defined in terms of activities) beta 0 = 0.78 +/- 0.08. The formation of analogous KOH 0 or CsOH 0 ion-pairs under the experimental conditions applied is negligible. Assuming the formation of the NaAl(OH)4 (0) ion-pair is not necessary for modeling the experimental findings, as its formation causes only secondary effects on the potentiometric patterns. It has also been shown that all experimental data can be interpreted quantitatively if the formation of the doubly charged dimeric aluminate species is included in the calculation of the changes in the mean activity coefficients. The formation constant of the aluminate dimer could not be estimated purely from the H2/Pt potentiometric data but a lower limit for its formation constant (defined in terms of activities) has been derived. These conclusions are in full congruency with those derived from the Raman spectra of solutions with similar concentrations, so the two independent experimental methods lead to the same set of chemical species in highly concentrated alkaline aluminate solutions.

Autocatalysis and self-inhibition: coupled kinetic phenomena in the chlorite-tetrathionate reaction.

The initial rate of formation of chlorine dioxide in the chlorite-tetrathionate reaction changes in an unusual fashion. The formal kinetic order of both reactants varies over a very wide range. Moreover, chlorite ion behaves not just as a simple reactant, but also as a self-inhibitor. A five-step scheme, derived from an eight-step mechanism, is proposed in which the autocatalytic formation of HOCl plays a central role in accounting for this kinetic behavior.