Series Of pH Values Research Articles

Intrinsic affinity of protein – ligand binding by differential scanning calorimetry

Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein–ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.

Assessing Long-Term Variations of pH in Caucasian Lakes by Bioindication Method (Diatom Analysis)

The processes of possible long-term variations of pH in Caucasian lakes were analyzed based on a study of lacustrine sediments from five small lakes of the Western and Central Caucasus. The study was focused on diatom complexes examined in core columns of bottom sediments. The values of pH were reconstructed using the authors’ principle of unification of hydrological characteristics. The study used the results of isotopic dating. Series of numerical values of pH with lengths from 2000 to 130 years were constructed for the examined lakes. The reconstructed series of pH values show no appreciable changes in the lakes of the Western Caucasus and suggest pronounced processes of alkalinization in the lakes of the Central Caucasus.

Thermodynamic controls on the kinetics of microbial low-pH Fe(II) oxidation.

Acid mine drainage (AMD) is a major worldwide environmental threat to surface and groundwater quality. Microbial low-pH Fe(II) oxidation could be exploited for cost-effective AMD treatment; however, its use is limited because of uncertainties associated with its rate and ability to remove Fe from solution. We developed a thermodynamic-based framework to evaluate the kinetics of low-pH Fe(II) oxidation. We measured the kinetics of low-pH Fe(II) oxidation at five sites in the Appalachian Coal Basin in the US and three sites in the Iberian Pyrite Belt in Spain and found that the fastest rates of Fe(II) oxidation occurred at the sites with the lowest pH values. Thermodynamic calculations showed that the Gibbs free energy of Fe(II) oxidation (ΔG(oxidation)) was also most negative at the sites with the lowest pH values. We then conducted two series of microbial Fe(II) oxidation experiments in laboratory-scale chemostatic bioreactors operated through a series of pH values (2.1-4.2) and found the same relationships between Fe(II) oxidation kinetics, ΔG(oxidation), and pH. Conditions that favored the fastest rates of Fe(II) oxidation coincided with higher Fe(III) solubility. The solubility of Fe(III) minerals, thus plays an important role on Fe(II) oxidation kinetics. Methods to incorporate microbial low-pH Fe(II) oxidation into active and passive AMD treatment systems are discussed in the context of these findings. This study presents a simplified model that describes the relationship between free energy and microbial kinetics and should be broadly applicable to many biogeochemical systems.

Modeling of protein conformational fluctuations in p Ka predictions

A method is presented to account for conformational fluctuations of a protein in predicting the p K a values of its titrating groups. Conformations of the protein are generated by conventional molecular dynamics or Monte Carlo simulations, in which the protonations of the titrating groups are fixed. For each protein conformation, the electrostatic free energies required to add a proton charge to a titrating group while other groups are either unprotonated or protonated are calculated within a dielectric continuum model. These are used to determine the mean protonations of the titrating groups in the conformation at a series of pH values. The mean protonations are then used to determine the relative weight of the particular conformation with the titrating groups having all possible protonations. A conformationally averaged mean protonation for each titrating group is finally obtained by the weighted sum of the group’s mean protonations in all the conformations. This method is applied to yeast iso-1-ferricytochrome c. The predicted p K a values are in general agreement with experimental results.

Constructing a Proton Titration Curve from Ion-Step Measurements, Applied to a Membrane with Adsorbed Protein

A new measuring method is described for obtaining a proton titration curve. The curve is obtained from a microporous composite membrane, consisting of polystyrene beads in an agarose matrix, with lysozyme molecules adsorbed to the bead surface. The membrane is incorporated into a sensor system by deposition on a silicon chip with a pH-sensitive ion-sensitive field effect transistor (ISFET) located in the middle of a Ag/AgCl electrode. The actual measurement is performed by creating a stepwise change in the salt concentration of the bathing electrolyte (the ion step) and measuring the ISFET potential versus the Ag/AgCl electrode. This potential shows a transient change in the ion step, which indicates a transient pH change in the membrane. This procedure is repeated at a series of pH values. Equations are presented to calculate the proton titration curve of the membrane from the amplitude and duration of the measured transients. Measurements show qualitative agreement between the curves obtained and equilibrium titration experiments on the same system.

Identification of the ligand-exchange process in the alkaline transition of horse heart cytochrome c.

Magnetic-circular-dichroism (m.c.d.) spectra over the wavelength range 300-2000 nm at room temperature and at 4.2K of horse heart cytochrome c are reported at a series of pH values between 7.8 and 11.0, encompassing the alkaline transition. The effect of glassing agents on the e.p.r. spectrum at various pH values is also reported. Comparison of these results with spectra obtained for the n-butylamine adduct of soybean leghaemoglobin support the hypothesis that lysine is the sixth ligand in the alkaline form of horse heart cytochrome c. The m.c.d. and e.p.r. spectra of horse heart cytochrome c in the presence of 1-methylimidazole have also been examined. These studies strongly suggest that histidine-18, the proximal ligand of the haem, is the ionizing group that triggers the alkaline transition. Low-temperature m.c.d. and e.p.r. spectra are also reported for Pseudomonas aeruginosa cytochrome c551. It is shown that no ligand exchange takes place at the haem in this species over the pH range 6.0-11.3.

Hypothesis—A chemical mechanism for the biosynthesis of ATP involving ion-exchange reactions

Dissociation constants for Mg · ATP were determined by displacing ATP from Dowex-1 resin with magnesium. These constants were then used to analyze the kinetics of yeast mitochondrial ATPase, in terms of the concentrations of free magnesium and free ATP, at a series of pH values. Both Mg · ATP and hydroxide ions were found to compete with the binding of ATP to the enzyme. These results were interpreted, in terms of an ion-exchange model, to mean that the synthesis of ATP may require the utilization of both magnesium and hydroxide ions for the dissociation of ATP from the enzyme as Mg · ATP. The concentrations of Mg and hydroxide required to compete with ATP were both found to be about three orders of magnitude greater than those required to form products, indicating that magnesium and hydroxide ions can contribute about 8 kcal of energy when ATP is synthesized.

X-ray photoelectron spectroscopic (xps) surface characterization of cobalt on the surface of kaolinite

The adsorption of cobalt(II) on kaolinite was studied at selected pH values in the range 3–10. The chemical nature of adsorbed cobalt was characterized by XPS spectral features, binding energy measurements, Co 2 p satellite characteristics, and L 3VV Auger (X-ray induced) kinetic energy measurements. That the Co 2 p spectral features are not identical for samples prepared at a series of pH values, provides experimental evidence that the chemical nature of the cobalt-clay interaction changes with pH. The presence of satellite features and the Co 2 p 1 2 -Co 2 p 3 2 separation indicate Co(H) on kaolinite. The Co 2 p 3 2 binding energies decrease from 782.2 eV for kaolinite treated at pH 3 to 781.0 eV for the pH 10 sample. The binding energies for pH 7.8–10.00 samples are in close agreement with that for authentic Co(OH) 2. Satellite structure in transition metal 2 p XPS spectra is due to simultaneous excitation of electrons and corresponds to charge transfer processes from ligand to the metal. The satellite intensity and energy separation from the main photopeak are related to the extent of covalent bonding in the metal compound. Intense satellite peaks were observed for high spin covalent Co(II) compounds. The ratio of the Co 2 p satellite peak intensity to the main Co 2p photopeak decreases from 2.2 for the pH 5 sample to 1.21 for the pH 10 sample. The large satellite intensity ratio at low pH values suggests that Co(II) is more covalently bound to the clay at pH 5 than at higher pH values. The Co L 3VV Auger kinetic energy results also show a variation for samples prepared at different pHs. In model systems Auger transitions occur at high kinetic energies for elements in environments of high electron density. The L 3VV Auger transitions occur at 770.3 eV for the pH 6 sample and 768.6 eV for samples prepared above pH 7.8. These results are consistent with the interpretation of the satellite intensity ratios and binding energy measurements and support the notion of covalent Co(II)- clay bonding at low pH values.

Kinetic Studies on Soluble and Insoluble Urokinases

A water-insoluble urokinase (ins-UK) was prepared by covalent coupling to an electrostatically neutral polyacrylamide derivative. The esteratic activity retained by the bound enzyme is about 70 percent of that of the soluble urokinase (UK). Comparative kinetic studies of these two forms of the enzyme were undertaken on lysine esters: N-alpha-acetyl-L-lysine-methyl ester (ALEe) and N-alpha acetylglycyl-L-lysine methyl ester (AGLMe). It was first observed that these substrates both exhibit a marked inhibitory effect toward soluble UK, whereas this phenomenon was less manifest with the insoluble form of the enzyme. Michaelis constants and maximal velocities measured at 33 degrees C, for UK and ins-UK, were identical when ALMe was used, but slightly different with AGLMe. Determination of initial velocities, at a series of pH values shows only minimal differences in the behavior of the soluble enzyme with respect to that of the insoluble form. However, over a range of temperatures, differing Km values for these two enzyme forms were obtained using AGLMe as the substrate. These last results suggest possible interactions between the substrate and the insoluble carrier of the enzyme.

The potential of the In3+/In electrode

The potentials of cells of the type In (s) |InCl 3(aq)(m1) , HCl (0.02 mol kg −1 ) | AgCl (s) |Ag (s) have been measured and compared with those of the nominal type In (s) |InCl 3(aq)(m1) |AgCl (s) |Ag (s) at a series of pH values. Our results show that the hydrolysis of InCl 3 is important at certain pH values and that a satisfactory estimate of E' 0 and (ϖ E' 0 /ϖ T ) p for the In 3+ /In couple can only be made if this hydrolysis does not interfere with the electrode reaction. In 3+ +3 e →In. Values of E' 0 are estimated and compared with earlier determinations.

The potential of the In 3+/In electrode

Summary The potentials of cells of the type In (s) |InCl 3(aq)(m1) , HCl (0.02 mol kg −1 ) | AgCl (s) |Ag (s) have been measured and compared with those of the nominal type In (s) |InCl 3(aq)(m1) |AgCl (s) |Ag (s) at a series of pH values. Our results show that the hydrolysis of InCl 3 is important at certain pH values and that a satisfactory estimate of E' 0 and (ϖ E' 0 /ϖ T ) p for the In 3+ /In couple can only be made if this hydrolysis does not interfere with the electrode reaction. In 3+ +3 e →In. Values of E' 0 are estimated and compared with earlier determinations.

The rennin-sensitive bond of bovine κ-casein

1. 1. The action of rennin on purified S-carboxymethyl- κ-casein was followed by measuring the decrease in pH, under disaggregating conditions, at a series of pH values within the range 6.0–9.5. 2. 2. This difference in pH was converted to the number of protons released by taking into account the buffering capacity of the protein at each pH. 3. 3. An ionizing group with an apparent p K of 7.6 was observed to be released by the action of rennin. It is concluded that this group is the new amino group formed from the proteolytic attack by rennin on the labile peptide bond linking para-κ-casein to the macropeptide.

The stability of the protective antigen and histamine-sensitizing factor of Bordetella pertussis to acid and alkali.

Pertussis sonic lysates were adjusted to a series of pH values between 3.0 and 11.0, held for 1 hour at room temperature, and then returned to neutrality and assayed for residual mouse protective antigen (MPA) and histamine-sensitizing factor (HSF) activities. The percentage in-activation of MPA at pH 3.0, 10.0, and 11.0 was respectively 30, 18, and 40 but only the last value is significantly different from the pH 7.2 control. The percentage inactivation of HSF in the same samples was 40, 29, and 75 respectively. Exposure to pH 11.0 had a significantly greater destructive effect on HSF than on MPA.These data may be of future value in helping to develop methods for purifying MPA, for they indicate that this factor is reasonably stable to pH within a usefully wide range.

The pK of Specific Groups of Proteins: I. THE α-AMINO GROUP OF THE α CHAIN OF HUMAN CO-HEMOGLOBIN

Abstract The pH-dependent second order rate constant for the reaction of fluorodinitrobenzene with the α-amino group of the α chain of human CO-hemoglobin was determined at 25° and a series of pH values from 6 to 8. The pK of this group was calculated from these data to be 6.72 ± 0.03. The pH-independent second order rate constant (the rate constant for the uncharged amino group) was calculated to be 0.236 ± 0.009 m-1 sec-1. The pK values and the pH-independent second order rate constants of the α-amino groups of the peptides valylleucine and valylleucine amide were determined by the same method. For valylleucine the pK was found to be 7.90 ± 0.03 and the pH-independent second order rate constant was 0.062 ± 0.003 m-1 sec-1. These constants for valylleucine amide were found to be 7.65 ± 0.03 and 0.061 ± 0.002 m-1 sec-1. The pK values of the α-amino groups of the peptides determined by this kinetic method were found to be identical with the values independently determined by a potentiometric method. The sum of the rate constants for the reaction of fluorodinitrobenzene with all of the reactive groups of human CO-hemoglobin was also determined at a series of pH values. These data indicate that, in addition to the α-amino group of the α chain, at least two other groups react with this reagent under the conditions employed. One group or set of groups with a pH-independent rate constant lower than that of the α-amino group of the α chain appears to have a pK below 8, while another group or set of groups with a high pH-independent rate constant appears to have a pK above 9.

Titration curves of colloidal surfaces. II. Connective tissues

The colloidal charge density of certain connective tissues was measured in vivo by the method of diplaced Donnan potentials at a series of pH values from 7.4 to 2.2 to yield titration curves of the colloidal surfaces. Tendon, dermis, and cartilage of rat and rabbit were studied. Hydrogen ions and other cations including lysine, glucosamine, and protamine displace the titration curves at all pH levels through the formation of complexes or by the neutralization of negatively charged groups. The equilibrium constants and standard free energies of the reactions were estimated. Anions such as picric acid displace the titration constants toward higher pH levels, with a resulting uptake of hydrogen ions at the surface. Complex formation strongly influences the distribution of inorganic ions in tissues. Differences in the shape of the titration curves are explained in terms of internal electrostatic bonds and the distribution of electric charge.

NON‐SYMBIOTIC DEVELOPMENT OF SEEDLINGS OF CALLUNA VULGARIS

New PhytologistVolume 32, Issue 2 p. 115-127 Free Access NON-SYMBIOTIC DEVELOPMENT OF SEEDLINGS OF CALLUNA VULGARIS LEWIS KNUDSON, LEWIS KNUDSONSearch for more papers by this author LEWIS KNUDSON, LEWIS KNUDSONSearch for more papers by this author First published: June 1933 https://doi.org/10.1111/j.1469-8137.1933.tb07002.xCitations: 13AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Knudson, Lewis. Physiological study of the symbiotic germination of orchid seeds. Bot. Gaz. 79, 345– 79. 1925. 2 Knudson, Lewis Seed germination and growth of Calluna vulgaris. New Phytol. 28, 369– 76. 1929. 3 Rayner, M. C. Obligate symbiosis in Calluna vulgaris. Ann. Bot. 29, 97– 133. 1915. 4 Rayner, M. C. The nutrition of Mycorrhiza plants: Calluna vulgaris. Brit. Journ. Exp. Biol. 2, 265– 92. 1925. 5 Rayner, M. C. Mycorrhiza: An account of non-pathogenic infection by fungi in vascular plants and bryophytes. New Phytol. Reprint, 15, 1– 246. 1927. 6 Rayner, M. C. Seedling development in Calluna vulgaris. New Phytol. 28, 377– 85. 1929. 7 Wilson, J. K. Calcium hypochlorite as a seed steriliser. Amer. Journ. Bot. 2, 420– 7. 1915. Citing Literature Volume32, Issue2June 1933Pages 115-127 ReferencesRelatedInformation