Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods

The efficiency of eight extracellular polymeric substances (EPS) extraction methods was compared on two different activated sludges. Three chemical methods (EDTA, formaldehyde + NaOH, glutaraldehyde), four physical methods (sonication, cation exchange resin, sonication + cation exchange resin, heating) and a control method (centrifugation alone) were tested.EPS quantities extracted were more greater for chemical methods than those for physical methods. For the chemical methods used EPS contamination due to extracting reagents was pointed out by infra-red analysis. The EPS extracted by physical methods can show a different qualitative composition with protein and carbohydrate as predominant compounds. This study therefore underlines that the choice of EPS extraction method should not only be limited to extraction yield and nucleic acid content but should also consider that the EPS solution may be contaminated by extracting reagents and/or be greatly modified by the extraction protocol.     

Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols

The characteristics of the extracellular polymeric substances (EPS) extracted with nine different extraction protocols from four different types of anaerobic granular sludge were studied. The efficiency of four physical (sonication, heating, cationic exchange resin (CER), and CER associated with sonication) and four chemical (ethylenediaminetetraacetic acid, ethanol, formaldehyde combined with heating, or NaOH) EPS extraction methods was compared to a control extraction protocols (i.e., centrifugation). The nucleic acid content and the protein/polysaccharide ratio of the EPS extracted show that the extraction does not induce abnormal cellular lysis. Chemical extraction protocols give the highest EPS extraction yields (calculated by the mass ratio between sludges and EPS dry weight (DW)). Infrared analyses as well as an extraction yield over 100% or organic carbon content over 1 g g⁻¹ of DW revealed, nevertheless, a carry-over of the chemical extractants into the EPS extracts. The EPS of the anaerobic granular sludges investigated are predominantly composed of humic-like substances, proteins, and polysaccharides. The EPS content in each biochemical compound varies depending on the sludge type and extraction technique used. Some extraction techniques lead to a slightly preferential extraction of some EPS compounds, e.g., CER gives a higher protein yield.     

Cd2+ activation of l-threonine dehydrogenase from Escherichia coli K-12

Homogeneous preparations of l-threonine dehydrogenase (l-threonine: NAD⁺ oxidoreductase, EC 1.1.1.103) from Escherichia coli K-12, after having been dialyzed against buffers containing Chelex-100 resin, have a basal level of activity of 10–20 units/mg. Added Cd²⁺ stimulates dehydrogenase activity approx. 10-fold; this activation is concentration-dependent and is saturable with an activation K_d = 0.9 μM. Full activation by Cd²⁺ is obtained in the absence of added thiols. The pH-activity profile of the Cd²⁺-activated enzyme conforms to a theoretical curve for one-proton ionization with a pK_a = 7.85. Mn²⁺, the only other activating metal ion, competes with Cd²⁺ for the same binding site. K_m values forl-threonine and NAD⁺ as well as the V_max for ‘demetallized’, Cd²⁺-activated, and Mn²⁺-activated threonine dehydrogenase were determined and compared.     

Thermodynamics of complexation of lanthanides by methoxybenzoates

The thermodynamic parameters of complexation of lanthanide cations by ortho-, meta- and para- methoxybenzoates have been measured using potentiometric and calorimetric techniques at 25 °C and an ionic strength of 0.10 M (NaClO₄). The values of logβ₁₀₁ correlate well with the ligand acid values of pK_a, reflecting the strongly ionic nature of the metal-ligand interaction. No evidence is found for extra charge polarization in these aromatic ligands due to the lanthanide complexation.     

UV-Vis and fluorimetric Al, Zn, Cd and Pb complexation studies of two 3-hydroxyflavones and a 3-hydroxythioflavone

The complexation of Al³⁺, Zn²⁺, Cd²⁺ and Pb²⁺ by the 3-hydroxyflavones: 3-hydroxy-2-(2-methoxyphenyl)-4H-1-benzopyran-4-one (H1) and 3-hydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one (H2), and by the 3-methoxythioflavone: 3-hydroxy-2-(2-methoxyphenyl)-4H-1-benzopyran-4-thione (H3) have been studied spectrophotometrically and fluorimetrically to determine the corresponding complexation constants, K_sp and K_fl, in 5:95 water:ethanol (v/v) solution for which [HClO₄] was either 10⁻² or 10⁻⁵ mol dm⁻³ and I = 0.10 mol dm⁻³ (NaClO₄) at 298.2 K. Complexation occurs dominantly through the deprotonated ligand for [Al(1)]²⁺ and [Al(2)]²⁺ for which log K_sp = 4.51 and 4.73, respectively, in 10⁻² mol dm⁻³ HClO₄ and 4.21 and 4.61 in 10⁻⁵ mol dm⁻³ HClO₄. For Pb²⁺ complexation by H1, H2 and H3 is characterized by log K_sp = 2.20, 2.57 and 3.22, respectively, in 10⁻² mol dm⁻³ HClO₄ and 4.70, 5.38 and 5.74 in 10⁻⁵ mol dm⁻³ HClO₄. Equilibrium mixtures of [Pb(H1)]²⁺ and [Pb1]⁺, [Pb(H2)]²⁺ and [Pb2]⁺, and [Pb(H3)]²⁺ and [Pb3]⁺ appear to be formed. Complexation of Zn²⁺ and Cd²⁺ by all three ligands was only detected in 10⁻⁵ mol dm⁻³ HClO₄. For Zn²⁺ complexation by H1, H2 and H3 log K_sp = 3.22, 3.74 and 4.46 and for Cd²⁺ the corresponding values are 2.39, 2.40 and 3.72 for Cd²⁺. Only [Al1]²⁺ and [Al2]²⁺ show significant fluorescence and are characterized by log K_fl = 6.30 and 7.49 in 10⁻² mol dm⁻³ HClO₄.     

Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K⁺-ATPase and the K⁺-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K⁺-stimulated ATPase, while K⁺-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K⁺-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K⁺-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K⁺-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K_m for p-nitrophenylphosphate unchanged for K_a for K⁺ increased. ATP, which is a potent inhibitor of K⁺-p-nitrophenylphosphatase activity in native membranes (K_I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K⁺-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K⁺-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H⁺-transport reaction.     

Lanthanide complexation with 1,3,5-triamino 2,4,6-trihydroxycyclohexane

The protonation constants of 1,3,5-triamino-2,4,6-trihydroxycyclohexane (taci), at 25 °C in I = 1.00 M (NaClO₄) were determined to be: pKa₁, 5.57 (0.08); pKa₂, 7.45 (0.02); pKa₃, 9.05 (0.04). The log of the stability constants, log β₃₀₂, at 25°C in I = 1.00 M (NaClO₄) for formation of were measured by potentiometry to be: Nd(III), 25.33 (0.09); Eu(III), 26.42 (0.06); Tm(III), 30.07 (0.10); Lu(III), 33.68 (0.07) ; Y(III), 28.59 (0.07). ¹H NMR spectra were consistent with formation of a single complex from pcH 6 to 10. Laser fluorescence measurements of the ⁷F_o-⁵D_o transition of Eu(III) complexed by taci indicated a single complexed species. The shift in this peak relative to that of Eu³⁺(aq) was significantly greater than the values reported for the complexes of other organic ligands with Eu(III). Luminescence lifetime measurements indicated two water molecules bound to each of the Eu(III) cations in the taci complex.     

Interaction of Aplysia brasiliana Myoglobin with Mn2+ and Cu2+ ions

Myoglobin of Aplysia brasiliana (MbApB) has been recently purified and characterized and it was shown that the amino acid content is quite different from other myoglobins. A large number of aromatic residues was observed together with the existence of a unique histidine at the proximal heme position. Because of the numerous differences in the amino acid sequence between MbApB and whale myoglobin, it was interesting to investigate the interaction of metal ions like Cu²⁺ and Mn²⁺ with MbApB. In the present work Cu²⁺ complexes with Met-MbApB were studied and show a pH transition between different forms of coordination as revealed by EPR measurements. At high pH the EPR spectrum shows the coordination of the metal to at least four nitrogens from ϵ-NH₃ lysine residues. At lower pH in the range 6.0–9.0 the copper binding site shows a pK change of some of the residues involved in metal coordination. Addition of one equivalent Cu²⁺ per protein does not alter the iron EPR signal. The manganese ion has one binding site in MbApB and a binding constant K_a = ( 11.5 ± 0.8) 10³M⁻¹. The binding of Cu²⁺ to MbApB is stronger than Mn²⁺, K_a^Cu2+ >K_a^Mn2+.     

The complexation of trivalent actinides by TMDTA

The stability constants in 0.10 M (NaCl) ionic strength solution for the formation of MTMDTA^?1 (M=Am, Cm, Bk and Cf; TMDTA = trimethylenedinitrilotetraacetate) were measured by solvent extraction. The values are 13.45 (Am), 13.79 (Cm), 14.36 (Bk) and 14.66 (Cf) and are much lower than expected from the σpK_a of the TMDTA acid. The data do not allow a definite interpretation of this decreased stability which may be due to a generalized weaker bonding or to failure to form the 6-membered

chelate ring.     

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na₂CO₃) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca²⁺). From the six extraction methods used, only the Na₂CO₃ extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca²⁺. The Ca²⁺-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.     

Role of extracellular polymeric substances in Cu(II) adsorption on Bacillus subtilis and Pseudomonas putida

The effect of extracellular polymeric substances (EPS) of Gram-positive Bacillus subtilis and Gram-negative Pseudomonas putida on Cu(II) adsorption was investigated using a combination of batch adsorption, potentiometric titrations, Fourier transform infrared spectroscopy. Both the potentiometric titrations and the Cu(II) adsorption experiments indicated that the presence of EPS in a biomass sample significantly enhance Cu(II) adsorption capacity. Surface complexation modeling showed that the pK_a values for the three functional groups (carboxyl, phosphate and hydroxyl) were very similar for untreated and EPS-free cells, indicating no qualitative difference in composition. However, site concentrations on the untreated cell surface were found to be significantly higher than those on the EPS-free cell surface. Infrared analysis provided supporting evidence and demonstrated that carboxyl and phosphate groups are responsible for Cu(II) adsorption on the native and EPS-free cells.     

The complexation chemistry of cyclohexaamyloses: Adducts with 1-adamantanecarboxylic acid and anion

A study is reported of complexation reactions of cyclohexaamylose (Cy) with 1-adamantanecarboxylic acid and its anion using conductometry, pH potentiometry, and ¹³C nmr spectrometry. Binary and ternary (2 mol Cy/mol substrate) complexes are detected with both the acid and anion, and standard entropies and enthalpies of complexation are determined from the temperature dependences of the formation constants for all except the very weak ternary complex with the anion. Both the ¹³C nmr results and the entropy of complexation confirm the earlier suggestion that the anion in binary complexation is structured with the adamantanyl group in proximity to, but not penetrating, the Cy cavity. However, a negative ΔS^o for formation of this complex is reported which casts doubt on an earlier proposal that the adamantyl binary complex binding mode involves an “apolar” mechanism accompanied by loss of solvated water molecules. Values are also reported for pK_a, ΔH^o, and ΔS^o for the aqueous dissociation of 1-adamantanecarboxylic acid.     

Protonation of kanamycin A: Detailing of thermodynamics and protonation sites assignment

Protonation of an aminoglycoside antibiotic kanamycin A sulfate was studied by potentiometric titrations at variable ionic strength, sulfate concentration and temperature. From these results the association constants of differently protonated forms of kanamycin A with sulfate and enthalpy changes for protonation of each amino group were determined. The protonation of all amino groups of kanamycin A is exothermic, but the protonation enthalpy does not correlate with basicity as in a case of simple polyamines. The sites of stepwise protonation of kanamycin A have been assigned by analysis of ¹H-¹³C-HSQC spectra at variable pH in D₂O. Plots of chemical shifts for each H and C atom of kanamycin A vs. pH were fitted to the theoretical equation relating them to pK_a values of ionogenic groups and it was observed that changes in chemical shifts of all atoms in ring C were controlled by ionization of a single amino group with pK_a 7.98, in ring B by ionization of two amino groups with pK_a 6.61 and 8.54, but in ring A all atoms felt ionization of one group with pK_a 9.19 and some atoms felt ionization of a second group with pK_a 6.51, which therefore should belong to amino group at C3 in ring B positioned closer to the ring A while higher pK_a 8.54 can be assigned to the group at C1. This resolves the previously existed uncertainty in assignment of protonation sites in rings B and C.     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H⁺ + K⁺)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg²⁺-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol P_i and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K⁺ (up to 0.69 ± 0.09 nmol P_i incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K⁺-stimulated ATPase and p-nitrophenylphosphatase activities and K⁺-sensitive phosphorylation: Mg²⁺-ATPase (μmol P_i/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K⁺-stimulated); Mg²⁺-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K⁺-stimulated); Mg²⁺-phosphorylation (nmol P_i/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K⁺). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H⁺ + K⁺)-ATPase in a still active structure.     

Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites

Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)₄G-NH₂] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M–S distances of 2.63 and 2.54 Å, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS₂ bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)₃ ^3? → M(II)(TRI LXC)₃ ^?. While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)₂(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)₃ ^?, the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)₂ ⁺ at pH < 5 followed by a two-proton dissociation step (pK _a2) yielding M(II)(TRI LXC)₃ ^?. Pb(II)(TRI LXC)(HTRI LXC)₂ ⁺ converts to Pb(II)(TRI LXC)₃ ^? at slightly lower pH values than the corresponding Cd(II)–peptide complexes. In addition, Pb(II) displays a lower pK _a of binding to the “d”-substituted peptide, (TRI L12C, pK _a2 = 12.0) compared with the “a”-substituted peptide, (TRI L16C, pK _a2 = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K _bind = 3.2 × 10⁷ M^?1) compared with that with TRI L16C (K _bind = 1.2 × 10⁷ M^?1) at pH > 8.     

Heme-linked ionization and chloride binding in intestinal peroxidase and lactoperoxidase.

Hog intestinal peroxidase and bovine lactoperoxidase exhibited similar spectral shifts upon pH alteration. From spectrophotometric titrations, it was found that there are hemelinked ionizations of pK_a = 4.75 in intestinal peroxidase and pK_a = 3.5 in lactoperoxidase. The apparent pK_a (pK_a′) increased with the increase in chloride concentration. The pK_a′ vs log[Cl^?] plots showed that the chloride forms complex with the acid forms of these enzymes with a dissociation constant (pK = 2.7). Although the dissociation constant (K_d) of the peroxidase-cyanide complexes is nearly independent of pH, cyanide competed with chloride in the acidic pH region. The slopes of logK_d vs log[Cl^?] were 1.0 for intestinal peroxidase and 0.5 for lactoperoxidase. The reaction of hydrogen peroxide with these peroxidases was also affected by chloride, similarly as the reaction with cyanide was. The results were explained by assuming that protonation occurs at the distal base and destroys the hydrogen bond between the base and a water molecule at the sixth coordinate position of the heme iron.     

Structural analysis of DNA complexation with cationic lipids

Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid–DNA interaction, while lipid-base binding and the stability of cationic lipid–DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microscopy were used to analyse lipid-binding site, the binding constant and the effects of lipid interaction on DNA stability and conformation. Structural analysis showed a strong lipid–DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K_Chol = 1.4 (±0.5) × 10⁴ M⁻¹, K_DDAB = 2.4 (±0.80) × 10⁴ M⁻¹, K_DOTAP = 3.1 (±0.90) × 10⁴ M⁻¹ and K_DOPE = 1.45 (± 0.60) × 10⁴ M⁻¹. The order of stability of lipid–DNA complexation is DOTAP>DDAB>DOPE>Chol. Hydrophobic interactions between lipid aliphatic tails and DNA were observed. Chol and DOPE induced a partial B to A-DNA conformational transition, while a partial B to C-DNA alteration occurred for DDAB and DOTAP at high lipid concentrations. DNA aggregation was observed at high lipid content.     

Rare earth complexation with 1,3,5-trideoxy-1,3,5-tris(2-hydroxybenzyl)amino-cis-inositol

The protonation constants of 1,3,5-trideoxy-1,3,5-tris(2-hydroxyl-benzyl)amino-cis-inositol (thci) in I = 1 M (NaClO₄) were determined to be: pK_a1 5.96 ± 0.03, pK_a2 7.21 ± 0.01, pK_a3 8.32 ± 0.07, pK_a4 8.95 ± 0.06. The solvent extraction studies were consistent with the formation of the Ln(thci)³⁺ and complexes. The log of the stability constants (log β₁ and log β₂) at 25 °C in 1 M (NaClO₄) at pH 4 for formation of these complexes are reported. Laser luminescence measurements of the ⁷F₀-⁵D₀ transition of Eu(III) complexed by thci indicated two species. The shifts in the peaks relative to that of Eu(aq)³⁺ were comparable to the values reported for other complexes of Eu(III) with organic ligands, but the intensities were greater. Luminescence lifetime measurements of the fluorescence spectra indicated that the complex has 5 inner sphere water molecules bound to the Eu(III) cation at pH 6.71-8.52. This was consistent with bidentate chelation of Eu(III) with each thci molecule. gaussian view energy calculations indicated bonding for M(III) to the amino and hydroxyl groups of the cyclohexanetriol and (2-hydroxybenzyl)amino moieties in the Ln(thci)³⁺ complex.     

Metal ion-biomolecule interactions. II. Methylmercuration of the deprotonated amino groups in adenine,guanine, and cytosine derivatives,and its relationship to amino group acidity

Proton nmr spectroscopic evidence is presented for methylmercury(II) binding to the deprotonated amino groups in adenosine, 9-methyladenine, guanosine, 1-methylguanosine, and cytidine under basic conditions. Except for the guanosine case, ¹H nmr spectra of the products from aqueous or ethanolic 1:1 mixtures of substrate and MeHgOH are consistent with methylmercuration of the deprotonated amino groups. Guanosine undergoes initial binding of MeHg to N₁, and a second equivalent of MeHgOH is necessary to effect amino binding. The nmr spectra of the complexed adenine derivatives suggest that different geometrical isomers exist in (CD₃)₂SO solution, reflecting the partial double bond character of the C₆N bond in these systems. Using a correlation relating the magnitude of the ¹⁹⁹Hg-¹H coupling constant (J) for MeHg-ligand complexes with the ligand pK_a (J = ?3.88 pK_a + 248.5, extending over 13 pK units, based on a variety of N and O donor ligands), estimates (± 0.3 pK unit) of the pK_as of the amino groups of the above substrates have been made. In this way, pK_a values of 15.5 (cytidine), 17.0 (adenosine and 9-methyladenine), 15.1 (guanosine), and 14.9 (1-methylguanosine) are obtained. In the cases where comparisons with literature pK_a data can be made, good agreement is found.