Alkaline Titrations of poly(dG-dC)·poly(dG-dC): Microemulsion Versus Solution Behavior

Abstract

PolyGC was titrated with a strong base in the presence of increasing concentrations of NaCl (from 0.00 to 0.60M) either in water solution or with the polynucleotide solubilized in the aqueous core of reverse micelles, i.e., the cationic quaternary water-in-oil microemulsion CTAB/n-hexane/n-pentanol/water. The results for matched samples in the two media were compared. CD and UV spectroscopies and, for the solution experiments, pH measurements were used to follow the course of deprotonation. In both media the primary effect of the addition of base was denaturation of the polynucleotide, reversible by back-titration with a strong acid.

In solution, the apparent pK_a of the transition decreases with increasing the salt concentration and a roughly linear dependence of pK_a on p[NaCl] has been found. A parallel monotonic decay with ionic strength has been found in solution for R_OH, defined as the number of hydroxyl ions required per monomeric unit of polyGC to reach half-transition. By contrast, in microemulsion, R_OH has been found to be independent of the NaCl concentration (and 10 to 50 times lower than in solution). This result is proposed as an indirect evidence of the independence of pK_a on the salt concentration in microemulsion, where the pH cannot be measured. A sort of buffering effect of the positive charges on the micellar wall and of their counter-ions on the ionic strength could well explain this discrepancy of behavior in the two media.     

Notice

¹⁷O NMR studies of ¹⁷O enriched water solutions containing superoxide dismutase have been performed between pH 7.5 and 11.7. Whereas T₁ measurements do not reveal any interaction between ¹⁷O and the paramagnetic copper center, the linewidth results appreciably increased with increasing pH with an apparent pK_a of 11.3. Comparison with ¹H NMR relaxation studies allows to interpret the present data as due to binding to the copper ion of an OH^? anion at high pH. The binding position should be of “equatorial” type, not involving the binding position of the coordinated water.     

Characterization of the isolated calcium-binding vesicles from the green alga Mougeotia scalaris,and their relevance to chloroplast movement

The calcium-binding vesicles from the green alga Mougeotia scalaris were isolated and characterized after staining in vivo by neutral red or rhodamine B. They were found to possess, a protonated group with a pK_a-9.9, typifying phenolic hydroxyl groups; upon titration, both, phenolic compound(s) and vital dye were concomitantly released from the vesicular matrix. A shift in peak absorbance from 450 nm to 540 nm of the vitally stained vesicles indicated that the neutral form of neutral red was bound to the vesicular, matrix as an intermediate form, stabilized via intermolecular hydrogen bonds to the phenolic compound(s). Up to 8.5.10⁹ dye molecules were calculated to be adsorbed to a mean-size vesicle. Analysis of Langmuir adsorption isotherms, indicated that there were two binding sites each for both neutral red and rhodamine B. The isolated vesicles were devoid of calcium, probably because vesicular calcium, bound to the vesicle matrix, was displaced upon dye binding. Dye adsorption to the vesicles in vivo results in substantial inhibition of the reorientational movement of the Mougeotia chloroplast and is explained by dye-mediated disorder of the cellular calcium homoeostasis.Abbreviations NR neutral red - RB rhodamine B - SDS sodium dodecyl sulfate This paper is part of the Ph.D. thesis of F. Grolig at Justus-Liebig-Universität Giessen, FRG     

A ketopentyl transition state analog for acetylcholinesterase.

4-oxo-N,N,N-trimethylpentanaminium chloride is a competitive inhibitor of eel acetylcholinesterase with K_I = 8 × 10^?6 M at 25°, 0.1 M NaCl, 0.04 M MgCl₂, pH 7.5. Its binding decreases at low pH with pK_a = 6.0. N,N,N-trimethylpentanaminium bromide has K_I = 4 × 10^?4 M under the same conditions. Its binding also decreases with pH with pK_a = 5.35. Comparison with literature data indicates that the ketone binds much more strongly than substrates and that its binding shows the pH dependence expected for a transition state analog.     

Establishment of low extracellular pH is essential for uptake of the fluorescent anionic dye hydroxypyrenetrisulfonate by suspension-cultured carrot cells

Uptake of the fluorescent dye hydroxypyrenetrisulfonate by carrot (Daucus carota L.) suspension cells only occurs when the external (medium) pH falls to below 4.0. Uptake of the dye was shown to be inhibited by a range of compounds, including the ‘Good’ buffers MES, HEPES, HEPPSO and HEPPS, the growth medium component coconut water (CW) and probenecid, an organic anion translocator inhibitor. Tris(hydroxymethyl)aminomethane stimulated transport. Buffer effects were not correlated with pK_a values. Inhibitors of dye uptake (MES, probenecid, CW) also inhibited medium acidification by the cells and Tris stimulated acidification and uptake. Uptake of HPTS correlated strongly with external pH and was restored when external pH was experimentally reduced to below 4.0 even in the presence of inhibitors. This suggests that inhibitors of HPTS uptake at the plasma membrane act primarily by preventing the establishment of a low external pH required for transport. The implications of this on the mechanism of HPTS transport are discussed.     

Electrostatics of folded and unfolded bovine β-lactoglobulin

We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine β-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5–9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pK _a value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pK _a value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH.     

Anion interactions with Na,K-ATPase: simultaneous binding of nitrate and eosin

Nucleotide binding affinity to Na,K-ATPase is reduced by a number of anions such as nitrate and perchlorate in comparison with affinity in the presence of chloride (all with sodium as the cation). The reduction correlates with the position of these anions in the Hofmeister series. Transient kinetic experiments using the fluorescent dye eosin—which binds to the nucleotide site of the Na,K-ATPase—show that simultaneous anion binding, exemplified with nitrate, and eosin binding is possible. The effect of nitrate on eosin binding is reflected in a decreased binding-rate constant and an increased dissociation rate constant, leading to a decreased equilibrium binding constant for eosin. Since eosin binding is analogous with nucleotide binding to Na,K-ATPase, the results suggest the simultaneous presence of nucleotide and anion binding sites.Abbreviations E₁ the protein conformation in Na⁺ - E₂ the enzyme conformation in K⁺ - Eo eosin (tetrabromofluorescein) - F fluorescence - I ionic strength - k_i rate constant - K_i equilibrium dissociation constant - K_i,0 equilibrium dissociation constant at zero ionic strength - N nitrate - z_i net charge - charge product z_i·z_j     

Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, erythrosin B, tetrachlorofluorescein, spectroscopic characterization of pure dyes, association of eosin Y

Analytically pure samples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependent on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimers D in monomers M at 293 K, pH = 12, is K21 = 2,9 X 10(-5) M; of the tetramers Q in dimers D K42 = 2,4 X 10(-3) M. From the experimental spectra of eosin solutions at various concentrations, pH = 12, and the equilibrium constants K21, K42 the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, VM = 19300 cm-1, epsilon M = 1,03 X 10(5) M-1 cm-1; D also one absorption band, VD = 19300 cm-1, epsilon D = 1,74 X 10(5) M-1 cm-1; Q two absorption bands, VQ1 = 19100, VQ2 = 20200 cm-1, epsilon Q1 = 1,65 X 10(5), epsilon Q2 = 1,96 X 10(5) M-1 cm-1. The absorption spectrum of the dimers is discussed by quantum mechanics.     

The role of the anionic groups in the receptor binding of interleukin-8 antagonists

Summary In an effort to determine the role of the acidic group in the receptor binding ofN-(2-hydroxy-4-nitrophenyl)-N′-(phenyl) urea, an interleukin-8B receptor antagonist, its binding and that of several analogs was measured as a function of pH. These titrations indicate that these ureas bind most strongly in their anionic form. Studies of antagonists, with different acidities, demonstrated that the greatest change in binding of each urea occurred around the pK _a of the compound being examined. The studies suggest that the increase in binding of the antagonists at higher pH is a result of the increased negative charge on the compounds rather than the effects of pH on the receptor or radioligand.     

Root exudates from sunflower (Helianthus annuus L.) show a strong adsorption ability toward Cd(II)

Abstract

Cd(II) adsorption of root exudates from sunflower (Helianthus annuus L.) seedling was investigated by Cd ion-selective electrode, Fourier Transform Infrared spectroscopy, and fluorescence spectroscopy. Root exudates from Helianthus annuus L. had strong adsorption ability toward Cd(II). The adsorption process was pH-dependent and the maximum adsorption capacity, 150.8 mg g^?1, was observed at pH 7.0. Root exudates had pK _a1 at 4.7 for carboxyl and pK _a2 at 9.2 for phenolic, and amino groups. The aliphatic and aromatic (C?H) groups, amide III group, and the C (=O)?O and sulfonate groups were responsible for Cd(II) adsorption. The excitation emission matrix fluorescence spectroscopy showed protein-like substances participated in Cd adsorption and formed strong complexes, with conditional stability constants of 4.70 and 4.32, which is a little lower than that determined by potentiometric methods, 5.13. The strong Cd complexing ability of root exudates implies that root exudates may significantly affect mobility, toxicity, and phytoavailability of Cd. Cd binding of root exudates may be attributed to its interaction with the proteins, polysaccharides, and phenolic compounds in root exudates.     

Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains

The effects of NaCl stress on cell area and intracellular pH (pH_i) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30–35%), it caused different pH_i changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pH_i homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pK_a (pK_a,i) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pK_a,i close to its pH_i homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pK_a,i to its pH_i homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pH_i were mainly due to hyperosmotic stress and not ionic stress.     

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein

Many macromolecular interactions, including protein‐nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature‐dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, ΔH_obs, and accounts for as much as a half of the observed heat capacity change, ΔC_p. However, there is still a substantial ΔC_p associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)₃₄ to SSB over a broad pH range (pH 5.0–10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25°C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)₃₄ binding over this entire pH range, with contributions from at least three sets of protonation sites (pK_a1 = 5.9–6.6, pK_a2 = 8.2–8.4, and pK_a3 = 10.2–10.3) and these protonation equilibria contribute substantially to the observed ΔH and ΔC_p for the SSB‐dT(pT)₃₄ interaction. The contribution of this coupled protonation (∼ −260 to −320 cal mol⁻¹ K⁻¹) accounts for as much as half of the total ΔC_p. The values of the “intrinsic” ΔC_p,0 range from −210 ± 33 cal mol⁻¹ °K⁻¹ to −237 ± 36 cal mol⁻¹K⁻¹, independent of [NaCl]. These results indicate that the coupling of a temperature‐dependent protonation equilibria to a macromolecular interaction can result in a large negative ΔC_p, and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand‐macromolecular interactions, as well as protein folding. Proteins 2000;Suppl 4:8–22. © 2000 Wiley‐Liss, Inc.     

The role of the anionic groups in the receptor binding of interleukin-8 antagonists

In an effort to determine the role of the acidic group in the receptor binding of N-(2-hydroxy-4-nitrophenyl)-N-(phenyl) urea, an interleukin-8B receptor antagonist, its binding and that of several analogs was measured as a function of pH. These titrations indicate that these ureas bind most strongly in their anionic form. Studies of antagonists, with different acidities, demonstrated that the greatest change in binding of each urea occurred around the pK_a of the compound being examined. The studies suggest that the increase in binding of the antagonists at higher pH is a result of the increased negative charge on the compounds rather than the effects of pH on the receptor or radioligand.     

Hydroxyl ion movements across the human erythrocyte membrane. Measurement of rapid pH changes in red cell suspensions

A stopped flow rapid reaction apparatus capable of following changes of ±0.02 pH unit in 0.1 ml of solution in less than 0.005 sec has been developed, utilizing a commercially available pH-sensitive glass electrode. Using this instrument, extracellular pH at 37°C was followed from less than 0.025 sec to 300 sec after mixing equal volumes of the following CO₂-free solutions: (A) normal human red cells, washed three times and resuspended in 150 mM NaCl at pH 7.2 with a hematocrit of 18%; and, (B) 150 mM NaCl adjusted with HCl or NaOH to pH 2.1 to pH 10.3. A minimum of 2 ml of mixture had to flow through the electrode chamber to ensure complete washout. The mixing process produced a step change in the pH of the extracellular fluid, after which exchanges across the red cell membrane and buffering by intracellular hemoglobin caused it to return toward pH 7.2 with an approximately exponential time course. Under the assumption that pH changes after mixing represent exchanges of hydroxyl for chloride ions across the cell membrane, hydroxyl ion permeabilities (P _OH ^- in cm/sec) were calculated and found to vary from 2 x 10^-4 at pH 9 to 4 x 10^-1 at pH 4 according to the empirical relationship P _OH ^- = 170 exp (-1.51 pH). The form of the dependence of P _OH ^- on extracellular pH does not appear compatible with a simple fixed charge theory of membrane permselectivity.     

Functionally-distinct proton-binding in HERG suggests the presence of two binding sites

HERG (Human ether-à-go-go-related gene) potassium channels are crucial for cardiac action potential repolarization. HERG channels are also found in neuronal and tumor cells. The effect of pH₀ on HERG is of clinical significance because of changes in pH during myocardial ischemia, inflammation, and respiratory alkalosis. We present evidence for the presence of multiple proton binding sites in HERG. Extracellular protons bind rapidly and reversibly to affect both activation and deactivation. However, these effects occur in two distinct pH₀ ranges. The deactivation rate has a pK_a of 6.76±0.02 compared to pK_a of 5.50±0.02 for changes in current suppression, which suggests the presence of at least two proton binding sites on HERG with functionally distinct properties.     

Determination of rate constants for nucleotide dissociation from Na,K-ATPase.

A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP.     

Esters of indole-3-acetic Acid from Avena seeds

The present studies showed that about 80% of the indole-3-acetic acid extractable from Avena kernels by aqueous acetone was esterified to polymers precipitable by ammonium sulfate and ethanol or acetone. The polymers were positively charged, being adsorbed to cation exchange columns at a pH of 3, or below, and eluted at a pH greater than 4. The polymers were heterogeneous with respect to size, about 5,000 to 20,000 daltons, and charge, exhibiting apparent pK_a values of 4.2 and 4.7. The polymer fractions contained esterified IAA, anthrone-reactive material that liberated glucose upon acid hydrolysis, phenolic compounds, and peptidic material with a high proportion of hydrophobic amino acids. Since the esterified IAA was unstable, establishing polymer purity was not possible, and the designation IAA-glucoprotein fraction was adopted.     

Potentiometric measurement of stability constants of complexes between fulvic acid carboxylate and Fe3+

The stability constants of complexes formed between iron (III) and fulvic acid extracted from organic manures and wastes such as urban domestic sewage sludge, farmyard manure, poultry manure and sulfitation pressmud were investigated by the potentiometric titration method in an ionic medium of 0.1 M KNO₃ at 25±1 °C. A modification of the Katchalsky's model was employed for the estimation of stability constants. The displacement of the titration curves due to presence of Fe³⁺ in FA solutions formed the basis of calculations. The weak acidic property of fulvic acids due to carboxyl groups resulted in buffering over a wide range of pH; fulvic acids were completely neutralized in the pH range of 7.00–8.85. Apparent dissociation constants (pK_APP) of weakly acidic carboxyl groups were a direct function of degree of dissociation (α_L) in the mid-range of titration curves but were non-linear at high and low α_L values. The stability constants for formation of Fe–FA complexes (log β_Fe) calculated from the titration data were in the range of 5.64–7.55, depending upon α_L and electrostatic properties of fulvic acids. The relatively high stability constants of Fe–FA complexes in comparison to those with other competing cations suggest that the Fe–FA complexes are relatively stable in a soil environment. This revised version was published online in June 2006 with corrections to the Cover Date.     

CERTAIN EFFECTS OF SALTS ON THE PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA

The effect of various substances on living cells may be advantageously studied by exposing them to such substances and observing their subsequent behavior in solutions of a basic dye, brilliant cresyl blue. The rate of penetration of the basic dye, brilliant cresyl blue, is decreased when cells are exposed to salts with monovalent cations before they are placed in the dye solution (made up with borate buffer mixture). This inhibiting effect is assumed to be due to the effect of the salts on the protoplasm. This effect is not readily reversible when cells are transferred to distilled water, but it is removed by salts with bivalent or trivalent cations. In some cases it disappears in dye made up with phosphate buffer mixture, or with borate buffer mixture at the pH value in which the borax predominates, and in the case of NaCl it disappears in dye containing NaCl. No inhibiting effect is seen when cells are exposed to NaCl solution containing MgCl₂ before they are placed in the dye solution. The rate of penetration of dye is not decreased when cells are previously exposed to salts with bivalent and trivalent cations. The rate is slightly increased when cells are placed in the dye solution containing a salt with monovalent cation and probably with bivalent or trivalent cations. In the case of the bivalent and trivalent salts the increase is so slight that it may be negligible.     

Acceptor and donor-side interactions of phenolic inhibitors in Photosystem II

Certain phenolic compounds represent a distinct class of Photosystem (PS) II Q_B site inhibitors. In this paper, we report a detailed study of the effects of 2,4,6-trinitrophenol (TNP) and other phenolic inhibitors, bromoxynil and dinoseb, on PS II energetics. In intact PS II, phenolic inhibitors bound to only 90-95% of Q_B sites even at saturating concentrations. The remaining PS II reaction centers (5-10%) showed modified Q_A to Q_B electron transfer but were sensitive to urea/triazine inhibitors. The binding of phenolic inhibitors was 30- to 300-fold slower than the urea/triazine class of Q_B site inhibitors, DCMU and atrazine. In the sensitive centers, the S₂Q_A⁻ state was 10-fold less stable in the presence of phenolic inhibitors than the urea/triazine herbicides. In addition, the binding affinity of phenolic herbicides was decreased 10-fold in the S₂Q_A⁻ state than the S₁Q_A state. However, removal of the oxygen-evolving complex (OEC) and associated extrinsic polypeptides by hydroxylamine (HA) washing abolished the slow binding kinetics as well as the destabilizing effects on the charge-separated state. The S₂-multiline electron paramagnetic resonance (EPR) signal and the ‘split’ EPR signal, originating from the S₂Y_Z state showed no significant changes upon binding of phenolic inhibitors at the Q_B site. We thus propose a working model where Q_A redox potential is lowered by short-range conformational changes induced by phenolic inhibitor binding at the Q_B niche. Long-range effects of HA-washing eliminate this interaction, possibly by allowing more flexibility in the Q_B site.