Average Dissociation Constant Research Articles

Physico‐chemical Properties of a DNA Binding Protein: Escherichia coli Factor H1

H1 factor is a heat-stable protein found in large amounts in Escherichia coli. In vitro, this protein has been found to stimulate transcription of lambda templates by E. coli DNA-dependent RNA polymerase. The subunit molecular weight of this factor has been re-estimated and found to be 15500 +/- 1000 in the presence of 2-mercaptoethanol. Crosslinking experiments performed with dimethylsuberimidate in the presence or absence of the DNA template indicate the presence of multiples of the 15500-Mr subunit up to the tetramer, the dimeric species being predominant. One cysteinyl residue per 15000-Mr subunit is labeled by 4-chloro-7-nitrobenzofurazan. This residue is not labeled if the factor is exposed to oxidizing conditions. In this case, three lysyl residues are titrated. H1 factor behaves as a DNA-binding protein. We have detected binding to DNA by two independent methods: displacement of a fluorescently labeled factor by the native protein and retention of radioactive DNA on millipore filters in the presence of the factor. Under our experimental conditions (high ionic strength, absence of magnesium ions), the saturation function of lambda plac DNA as well as of wild-type lambda DNA has been found to be non-cooperative. Saturation is reached when 300 +/- 30 molecules of dimeric factor are bound per lambda molecule, the average dissociation constant of the complex being 10nM. The dissociation time of the H1.DNA complex is less than 5 s at 37 degrees C. The binding of this factor lowers the affinity of native DNA for ethidium bromide. In the presence of this intercalating dye, the solubility of the complex decreases drastically.

The binding of 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate by human antithrombin III

The binding of 1-anilino-8-naphthalenesulfonate to human antithrombin III was studied by fluorescence enhancement of the fluorophor and fluorescence quenching of the protein emission. Two molecules of 1-anilino-8-naphthalenesulfonate were found to bind per antithrombin molecule with an average dissociation constant of 4.4·10 −5M. The binding of heparin to antithrombin was studied by ultraviolet difference spectroscopy. The stoichiometry of the heparin binding indicated 1.8 binding sites with an average dissociation constant of 4.3•10 −6M. Further the fluorometric competition experiments with 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate indicated two different classes of anion binding sites on the antithrombin molecule.

Prostaglandin E 1 specific binding in rhesus myometrium

Myometrial low speed supernatant prepared from non-pregnant rhesus uteri was incubated with 3H-Prostaglandin (PG)E 1 with or without addition of unlabelled prostaglandins. The uptake of 3H-PGE 1 was inhibited in a dose dependent fashion by PGE 2>PGE 1>PGA 1>PGF 2α=PGA 1>PGB 1=PGB 2≥PGD 2. PGE 1 metabolites inhibited 3H-PGE 1 binding in the following order: 13,14-dihydro-PGE 1>13,14-dihydro-15-keto-PGE 1=15-keto-PGE 1. The specific binding of 3H-PGE 1 and 3H-PGF 2α was similarly affected by the temperature and time of incubation. Equilibrium binding constants determined using rhesus uteri obtained during the luteal phase of the menstrual cycle indicate the presence of high affinity PGE 1 binding sites with an average (n=3) apparent dissociation constant of 2.2 × 10 −9M and a lower affinity PGE 1 binding site with a K d ≅ 1 × 10 −8M. No high affinity — low capacity 3H-PGF 2α sites could be demonstrated. Relative uterine stimulating potencies of some natural prostaglandins and prostaglandin analogs tested after acute intravenous administration in mid-pregnant rhesus monkeys corresponded with the PGE 1 binding inhibition of the respective compound. The uterine stimulating potencies of the prostaglandin analogs tested were: (15S)-15-methyl-PGE 2=16,16-dimethyl-PGE 2>17-phenyl-18,19,20-trinor-PGE 2>16 phenoxy-17,18,19,20-tetranor-PGF 2α=PGE 2=PGE 1=(15S)-15-methyl-PGF 2α>PGF 2α.

NaSO 4 ? ion pairs in aqueous solutions at pressures up to 2000 atm

Electrical-conductance measurements have been made at 25° C up to concentrations of 0.06M in aqueous solutions of Na2SO4 at atmospheric pressure and as a function of pressure up to 2000 atm. Calculations of the change of the dissociation constant for the NaSO 4 − ion pair with pressure indicate that the difference in partial molal volumes between products and reactants at infinite dilution is $$\Delta \bar V^ \circ \tilde = - 8.25{\text{ }}cm^3 /mole$$ . Using the equation of Davies, Otter, and Prue, we found the average dissociation constants for five concentrations between 0.005 and 0.06 moles/liter to be 0.097, 0.131, and 0.165 at 1, 1000, and 2000 atm, respectively, with a standard deviation of ±0.003. The atmospheric-pressure value is 0.080±0.016 over the entire concentration range from 0.00005 to 0.06 moles/liter, about half that obtained by Jenkins and Monk. This is consistent with a value ofK=0.077±0.006 recalculated from the limited-concentration-conductance work of Jenkins and Monk, with a value ofK=0.073 obtained by Fisher from data of Kurtze and Tamm on ultrasonic absorption in MgSO4−NaCl solutions, and withK=0.067 calculated by Fisher from the potentiometric data of Pytkowicz and Kester at high ionic strength. The relationship of this work to sound absorption and ion pairing in seawater is discussed. The predicted pressure dependence of the NaSO 4 − ion pair in seawater from this work differs substantially from earlier work by Kester and Pytkowicz and by Millero.

Prostaglandin E 1 specific binding in rhesus myometrium

Myometrial low speed supernatant prepared from non-pregnant rhesus uteri was incubated with 3H-Prostaglandin (PG) E 1 with or without addition of unlabelled prostaglandins. The uptake of 3H-PGE 1 was inhibited in a dose dependent fashion by PGE 2>PGE 1>PGA 1>PGF 2α=PGA 1>PGB 1=PGB 2≥PGD 2. PGE 1 metabolites inhibited 3H-PGE 1 binding in the following order: 13,14-dihydro-PGE 1>13,14-dihydro-15-keto-PGE 1=15-keto-PGE 1. The specific binding of 3H-PGE 1 and 3H-PGF 2α was similarly affected by the temperature and time of incubation. Equilibrium binding constants determined using rhesus uteri obtained during the luteal phase of the menstrual cycle indicate the presence of high affinity PGE 1 binding sites with an average (n=3) apparent dissociation constant of 2.2 × 10 −9M and a lower affinity PGE 1 binding site with a K d ≊ 1 × 10 −8M. No high affinity — low capacity 3H-PGF 2α sites could be demonstrated. Relative uterine stimulating potencies of some natural prostaglandins and prostaglandin analogs tested after acute intravenous administration in mid-pregnant rhesus monkeys corresponded with the PGE 1 binding inhibition of the respective compound. The uterine stimulating potencies of the prostaglandin analogs tested were: (15S)-15-methyl-PGE 2=16,16-dimethyl-PGE 2>17-phenyl-18,19,20-trinor-P GE 2>16 phenoxy-17,18,19,20-tetranor-PGE 2α=PGE 2=PGE 1=(15S)-15-methyl-PGE 2α>PGF 2α.

Studies on the fluorescence of 1-anilino-8-naphthalenesulfonate by the membranes of the sarcoplasmic reticulum.

The quantum yield of 1‐anilino‐8‐naphthalenesulfonate associated with native and lipid‐depleted sarcoplasmic vesicles is nearly identical. The slight increase of the average apparent dissociation constant of the interaction of this dye with lipid‐depleted vesicles is accompanied by a decrease in the number of binding sites for the dye. Oleic acid is a noncompetitive inhibitor of the anilino‐naphthalenesulfonate binding to native and lipid‐depleted sarcoplasmic vesicles. Rising temperatures decrease the quantum yield and increase the average apparent dissociation constant. The polarisation of the fluorescence of membrane‐bound anilino‐naphthalenesulfonate reaches relatively high values with a significant increment for lipid‐depleted sarcoplasmic vesicles. There is only a slight temperature dependence and no viscosity dependence for both native and lipid‐depleted vesicles. The polarisation is reduced by rising concentrations of anilino‐naphthalenesulfonate. Energy transfer measurements indicate that the dye is located very closely to the membrane‐bound tryptophan, suggesting a donor‐acceptor distance of 2 nm.

Progesterone "receptors" in the cytoplasm and nucleus of chick oviduct target tissue.

This report demonstrates that the chick oviduct, a specific target organ for progesterone, contains both cytoplasmic and nuclear macromolecules which bind progestins. These binding molecules can be clearly distinguished from transcortin by centrifugation through sucrose gradients of low ionic strength and by agarose gel filtration. The cytoplasmic progesterone-binding molecules also bind 5-alpha-pregnane-3,20-dione, but have significantly lower affinity for cortisol, estrone, or aldosterone. They are absent from blood and nontarget organs such as lung and spleen. The tissue-specific binding components appear to be heat-labile proteins with an average dissociation constant for progesterone of about 8 x 10(-10) M at 2 degrees C. These results are consistent with the identification of the progesterone-binding molecules as the functional hormone receptors. In further support of this concept is the finding that treatment of the chicks with estrogen coordinately induces a 20-fold increase in the number of progesterone-binding molecules and enhances the capacity of progesterone to induce avidin synthesis.A progesterone-"receptor" complex can be detected in both the cytoplasm and nuclei of oviduct tissue after an injection of [(3)H]progesterone to estrogen-treated chicks. By contrast, incubation of oviduct tissue with [(3)H]progesterone in vitro at 2 degrees C for 5 min leads to labeling of the cytoplasmic "receptor" only. Transfer of the "receptor"-steroid complex into the nucleus then appears to occur upon subsequent incubation in vitro at 37 degrees C. This observation suggests that the transfer of bound progesterone across the nuclear membrane may be an energy-requiring enzymatic process.

TISSUE CARBON DIOXIDE STORES: MAGNITUDE OF ACUTE CHANGE IN THE DOG.

Carbon dioxide washout curves were determined in hyperventilated dogs. Direct measurement of mixed venous carbon dioxide tension allowed calculation of changes in whole-body CO2 stores. The average whole-body CO2 dissociation constant in ten studies was 3.73 ml/kg mm. The limiting factor in reaching a new steady-state value was represented by a slow compartment in the washout curve. The average rate constant for this compartment was 0.062 min–1. The slowest compartment in this analysis has a 98% change in 1 hr, therefore the experimentally determined whole-body dissociation constant should closely approximate actual changes in tissue CO2 stores, excluding bone and fat.

Spectrophotometric determination of ruthenium

A new method for the spectrophotometric determination of ruthenium with anthranilic acid has been described. The reagent, at PH range 5.2–6, forms with ruthenium(III) a green complex having absorption maximum at 620 nm. The colour system obeys Beer's law for 1 to 20 ppm with an optimum concentration range of 4 to 16 ppm where the per cent relative error per 1% absolute photometric error is 3.2. Job and molar ratio methods indicate the formation of a 1∶2 complex between ruthenium(III) and the reagent and the average dissociation constant of the complex is 1.74 · 10−8 at 24° C.

Spectrophotometric determination of platinum o-phenylenediamine as a reagent

Summary The orange color complex formed by platinum(IV) with o-phenylenediamine at the p h range 3–4 forms the basis of sensitive colorimetric method for determination of the metal. The color complex has an absorption maximum at 450 mμ and obeys Beer's law for a platinum(IV) concentration of 1 to 12 p.p.m., the optimum concentration range being 4 to 12 p.p.m. where the per cent relative error per 1% absolute photometric error is 2.87. Sensitivity of the color reaction is 1 part of platinum per 59,000,000 parts of the solution. Continuous variation and molar ratio methods indicate that platinum(IV) forms with the reagent a 1 3n2 complex which has an average dissociation constant of 2.3.·10−8 at room temperature.

Spectrophotometric determination of osmium: Anthranilic acid as a reagent

Anthranilic acid has been found to be a highly sensitive reagent for the spectrophotometric determination of osmium(IV), osmium(VI) and osmium(VIII) at pH 6.0. In all three valence states the metal reacts with the reagent to form a dark-violet coloured compound, which shows maximum absorbance at 460 mμ. Osmium(IV) can be determined in the presence of diverse ions, if EDTA (disodium salt) is used as a masking agent. The optimun concentration range for the method is 2 to 6 p.p.m. of osmium(IV), where the % relative error per 1% absolute photometric error is only 2.8. By applying J ob's method of continuous variation, the molar ratio method and the slope ratio method, it was established that a 1 : 1 complex is formed between osmium(IV) and the reagent. The reaction of the reagent with osmium tetroxide or osmate indicates that the reagent is first oxidised and that then the reduced osmium(lV) forms a 1 : 1 complex with unoxidised excess reagent. The complex is stable in water and alcohol and has an average dissociation constant of 4.6 · 10 -5.

Spectrophotometric determination of osmium: Sulphanilic acid as a reagent

Sulphanilic acid has been found to be a very effective reagent for the spectrophotometric determination (if hexavalent and octavalent osmium in the pH range between 1.8 – 3.5. In these two valence states, the element forms a dark-violet complex with the reagent, the absorption maximum of the complex being at 490 mμ. As most of the other ions interfere in the determination, the element must be separated as osmic acid by nitric acid distillation. Beer's law is obeyed in the case of 0.5 to 9 p.p.m. of osmium (VIII) and l to 18 p.p.m. of osmium (VI); tin-optimum concentration ranges are from 2 to 8 p.p.m. for osmium(VIII) and from 4 to 16 p.p.m. for osmium(Vl). In these ranges, the % relative errors per 1% absolute photometric error are 3.02 for osmium (VIII) and 3.1 for osmium(VI). Application of the method of continuous variations and the molar ratio method indicates that in solution hexavalent osmium and the reagent form a 1:2. complex, with an average dissociation constant of 1.2 ⋯ 10 -7.

Spectrophotometric determination of palladium : Bismuthiol ii as an analytical reagent

A method for the spectrophotometric determination of palladium with bismuthiol 11 is described. The colour reaction is instantaneous and at 20–30° the system is stable for about 24 hours when maintained at a pH between 3 and 10. The colour system shows no sharp peak of maximum absorption but measurements can be made at any wavelength between 410 mμ and 430 mμ, preferably at 420 mμ. In this region the system obeys Beer's law at a palladium concentration of 0.4 μg to 80μg per ml. The sensitivity of the reaction is 0.0t6μg palladium per cm 2 (practical) or o 01 μg and 0.012μg of palladium per cm 2 at 410 mμ and 420 mμ respectively (Sandell). Acetone and methyl cellosolve stabilize the system. Excess of reagent and a large number of cations and anions are completely without effect. The composition of the complex in solution, which agrees well with that of the isolated compound, is Pd(C 8H 5N 2S 3) 2 with an average dissociation constant of 2.8 . ̄ 10 -12 at 25°.

Theory of the effect of counterion size upon titration behavior of polycarboxylic acids

AbstractThe effect of the ionic radius of counterions has been introduced into the dissociation theory of polyelectrolytes, using the familiar rod‐like model. Increments in the average dissociation constant pKa and slope n for polyacrylic acid with increasing size of the gegenion have been calculated and compared with data using potassium and some tetraalkylammonium hydroxides. Good agreement is encountered. The effects of changes in concentration of the polyacid and of the presence of neutral salt are also considered.

Peroxy titanium oxalate

1. The method of the preparation of the normal peroxy titanium oxalate TiO2C2O4, 3·5 H2O is described. If an excess of hydrogen peroxide is not maintained in the starting solution, substances with the molar compositions Ti2O3 (C2O4)2, 7 H2O and Ti1·5O2·5 (C2O4)1·5, 5·5 H2O are obtained. 2. It has been shown by the various physico-chemical studies that the substance TiO2C2O4, 3·5 H2O is a true peroxy complex, while the other two substances are mixtures of the peroxy complex and its deoxygenated product (basic oxalate TiOC2O4) in different proportions. 3. The keeping quality of the peroxy complex indicates that the complex loses the peroxy oxygen gradually, transforming itself into the basic oxalate. 4. Molecular weights of the three complexes described in (1) have been determined cryoscopically. 5. Conductivity of the aqueous solution of the normal peroxy complex gave the average dissociation constant K = 4·5 × 10-2. 6. Spectrophotometric investigations on the peroxy complex showed that the peroxy complex has the atomic ratio of Ti : O (peroxy) as 1:1 and the absorption maximum of the complex is at the wavelength 425 mµ. 7. Potentiometric titration of the aqueous solution of the complex against sodium hydroxide indicated the dibasic character of the complex in the solution. 8. Vapour pressure data for the normal peroxy complex gave the heat of dissociation equal to 15·4 K.cal./mole of water, indicating that the water associated with the complex is combined one. 9. Oxygen pressure for the normal peroxy complex varied from 0·12 to 0·30 mm. of mercury in the temperature range 2° to 45° C. 10. Dehydration studies on the normal peroxy titanium oxalate complex in vacuum showed that out of 3·5 moles of water, 1·5 moles are removed from the complex while 2 moles are retained with the complex. 11. Decomposition products obtained on heating the peroxy complex in vacuum are carbon dioxide, carbon monoxide, oxygen, water, titanium dioxide and carbon. 12. The complex has been assigned the structure Open image in new window based on various physico-chemical studies.

Polyelectrolytes. II. Poly‐4‐vinylpyridonium chloride and poly‐4‐vinyl‐N‐n‐butylpyridonium bromide

AbstractPoly‐4‐vinylpyridine was precipitated from a toluene solution of the monomer after addition of benzoyl Peroxide. The sample used in these experiments had a molecular weight of 77,000, (DP 730) as determined by osmotic pressure in alcohol solution, and an intrinsic viscosity of 0.36 in this solvent. The hydrochloride is water soluble and behaves like the salt of an extremely weak base with an over‐all average base dissociation constant of 0.011 × 10−9, about one thousandth that of 4‐ethylpyridine. The decrease in apparent base strength is ascribed to the chain structure which produces high local concentrations of pyridine groups, independent of the total concentration. By addition of butyl bromide to the polymer in nitromethane, a long chain quaternary ammonium salt is formed which is also water soluble. Its conductance is not sensitive to concentration down to about 0.003 N, after which the conductance rises sharply with further dilution. Dissociation, calculated by the classical Arrhenius method, indicates that a minimum of about 20% of the bromide ions are free, the remainder being associated by Coulomb attraction to the chainlike positive ion. As the dielectric constant of the solvent is decreased by addition of ethanol, the relative amount of free bromide ions decreases. The viscosity of the polyelectrolyte in aqueous or alcoholic solution, when plotted in the conventional way (ηsp/c versus c), approaches infinity at zero concentration instead of approaching a limiting constant linearly. A plot of $ {{\eta _{sp} } \mathord{\left/ {\vphantom {{\eta _{sp} } {\sqrt c }}} \right. \kern‐\nulldelimiterspace} {\sqrt c }} $ versus $ \sqrt c $ is, however, linear; this behavior is characteristic of strong electrolytes. The coefficient of the square root term in concentration for the polymer is larger, by orders of magnitude, than that for ordinary electrolytes. The coefficient of the linear term in the square root function is about equal to the intrinsic viscosity of the parent polymer.

Cellulose and Sodium Hydroxide

THE British Cotton Industry Research Association has recently issued two memoirs (reprinted in the Journal of the Textile, Institute, vol. 20, T. 373, 1929; and vol. 21, T. 225, 1930) in which S. M. Neale describes some work on the physical chemistry of cellulose. Regarding cellulose as a linked series of glucose residues with −OH and −O-groups as reacting points, it is shown that the behaviour in alkaline solutions can be explained by treating the −OH groups as sources of potential acidity, with their capacity for liberating hydrion governed by the law of mass action, so that an average dissociation constant can be assumed for the primary acid ionisation of cellulose in any state of complexity. In developing this idea it is necessary to employ the Donnan equation of membrane equilibrium to allow for the fact that the assumed cellulose ion is coherent and unable to diffuse. By assuming an approximate value of 2 × 10−14 for the dissociation constant it is possible to calculate approximately the osmotic swelling pressure of cellulose in solutions of caustic soda of any concentration. The calculated osmotic pressure curve is strikingly similar to the curve obtained by plotting the imbibition of water by regenerated cellulose (cellophane sheet) against the concentration of alkali in which it is placed, while the amounts of alkali taken up are shown to be consistent with the stoichiometric conversion of cellulose into the mono-sodium salt at high alkali concentrations, when allowance is made for the alkali imbibed in accordance with the Donnan equation.