Dissociation Constant

HIV-1 integrase is responsible for one of the key steps of the viral replication, integration of the viral cDNA into the host cell genome. Integration inhibition leads to complete block of the virus replication. In this study inhibition of integration by dimeric bisbenzimidazoles DBBI(7) with heptamethylene and DBBI(8) with tri(ethylene glycol) spacers was examined, and it was learned out that IC50 for DBBI(7) was about 0.03 microM, and IC50 for DBBI(8) was about 10 microM. By using cross-linking assays, it was shown that both compounds impeded a proper disposition of DNA-substrate at the active centre of integrase. Dissociation constants for complexes between either DBBI and DNA-substrate of integrase were determined. Calculated Kd values were 270 nM and 140 nM for complexes formed by DBBI(7) and DBBI(8), respectively. Therefore, inhibition of integration does not directly result from the binding of DBBIs with DNA. The dependence of initial rates of enzymatic reaction on the DNA-substrate concentration in presence of different concentrations of inhibitors was found, and inhibition constants were determined. All the data obtained allow us to suppose that the different inhibition activity of DBBI(7) and DBBI(8) results from the different mechanism of their binding: DBBI(7) is a competitive inhibitor of integrase whereas DBBI(8) is assumed to show a more complex mechanism of inhibition.

The pharmacological profiles of D1- and D2-like dopamine receptors were investigated for native brain receptors in the leopard frog, Rana pipiens, using direct binding assays, which characterize functional receptors rather than assess total receptor protein. We used homogenate assays of R. pipiens fore- and midbrains to determine, via saturation isotherms, that the dissociation constant, Kd, for (3)H-SCH-23390 binding to the D1-like receptors was 0.29 nM, and the maximal receptor density, Bmax, was 40 fmoles/mg protein. This compares with the more than 10-fold higher density of D1 sites in rat striatum. Specific binding for the D2-like receptors was measurable using these methods with (3)H-spiperone as the ligand. However, saturation of binding was not achieved. This contrasts with the > 400 fmoles/mg protein Bmax in rat striatum. Pharmacological profiles (rank order of potency of displacing drugs) for each receptor type were determined. We used non-radioactive SCH-23390, SKF-38393, sulpiride, and spiperone to displace (3)H-SCH-23390 and (3)H-spiperone at D1 and D2 receptors, respectively. Parallel displacement assays were performed with rat striatal controls. Results indicated that the relative rank order displacements in anuran dopamine receptors were characteristic of D1- and D2-like receptors. However, the rank orders were not identical to those in mammals. The rank order for affinity at D1-like receptors in both rats and frogs was SCH-23390 > SKF-38393 > spiperone > sulpiride. The rank order for affinity at D2-like receptors was spiperone > SCH-23390 > sulpiride > SKF-38393 in frogs, and spiperone > sulpiride > SCH-23390 > SKF-38393 in rats. SKF-38393 and spiperone had similar affinities for the 'D1' receptors in both species. SCH-23390 had a slightly lower affinity for the D1-like receptors in Rana, whereas sulpiride had a significantly lower affinity for Rana D1-like receptors compared to rat D1 receptors. In Rana D2-like receptors, spiperone and sulpiride were significantly less potent compared to rat. However, SCH-23390 and SKF-38393 were equally potent for the D2-like receptors in both species. The results indicate that amphibian brain dopamine receptors fall into two classes similar to the mammalian D1 and D2 subfamilies, but with binding characteristics slightly different from those typically described in mammals. This work represents the first pharmacological characterization of native brain dopaminergic receptors in an anuran amphibian. Because direct binding assays measure the initial aspect of the functional interaction between transmitter and receptor, these data provide an important complement to studies using cell expression systems.

A keratinase enzyme was isolated and purified from a feather-degrading culture of Aspergillus oryzae. Fractional precipitation of the crude enzyme with ethanol, acetone and ammonium sulfate yielded 21 fractions. The fraction obtained at 75–85% ammonium sulfate saturation ...

We have determined or redetermined the calcium dissociation constants of seven BAPTA-type buffers with KD's in the range from 0.4 microM to about 20 mM in 300 mM KCl. These include four newly synthesized ones: 5-nitro BAPTA; 5,5'-dinitro BAPTA; 5-methyl-5'-nitro BAPTA; and 5-methyl-5'-formyl BAPTA. Moreover, we tabulate dissociation constants or KD's for BAPTA and eleven BAPTA-type buffers, compare most of them with an empirical curve based upon so-called Hammett values, and predict KD's for several still unsynthesized but potentially valuable buffers.

The acid dissociation constant is an important physicochemical parameter of a substance, and knowledge of it is of fundamental importance in a wide range of applications and research areas. We present a critical review of published methods and approaches for the determination of acid dissociation constants, with a special emphasis on the pKa values of active pharmaceutical ingredients.

Enzymes utilize substrate binding energy both to promote ground state association and to selectively lower the energy of the reaction transition state.i The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the center of a 20 base-pair DNA target site, with the N-terminal domain of the enzyme making extensive binding interactions with the left (−) side of the target site and the similarly structured C-terminal domain interacting with the right (+) side.ii Despite the approximate two-fold symmetry of the enzyme-DNA complex, we find that there is almost complete segregation of interactions responsible for substrate binding to the (−) side of the interface and interactions responsible for transition state stabilization to the (+) side. While single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (−) DNA half-site almost exclusively increase KD and KM*, and those in the (+) half-site primarily decrease kcat*. The reduction of activity produced by mutations on the (−) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the utilization of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (−) side do so by modulating KM*, while redesigns with altered specificities on the (+) side modulate kcat*. Our results illustrate how classical enzymology and modern protein design can each inform the other.

The pK 1* and pK 2* of H2SO3 have been determined in NaCl solutions as a function of ionic strength (0.1 to 6 m) and temperature (5 and 25 °C). The extrapolated values in water were found to be in good agreement with literature data. The experimental results have been used to determine the Pitzer interaction parameters for SO2, HSO 3-and SO 3-in NaCl solutions. The resultant parameters for NaHSO3 and Na2SO3 were found to be in reasonable agreement with the values for NaHSO4 and Na2SO4. It, thus, seems reasonable to assume that the interactions of Mg2+ and Ca2+ with HSO 3-and SO 3-can be estimated from the values with HSO 4-and SO 4-until experimental values are available. Measurements of pK 1* and pK 2* in artificial seawater were found to be in good agreement with the calculated values using the derived Pitzer parameters. It is, thus, possible to make reasonable estimates of the activity coefficients of HSO 3-and SO 3-ions and pK 1* and pK 2* for the ionization of H2SO3 in marine aerosols.

The effect of single and binary solvents on electronic absorption spectra of 1,3-bis[(3-(2-pyridyl) pyrazol-1 ylmethyl)] benzene (1,3-PPB) was examined. 1,3-PPB has two absorption maxima (250 and 280 nm) due to pyridyl pyrazole and benzene systems. In order to explain the obtained results, UV absorption frequencies of the electronic transitions were correlated using a total solvatochromic equation by the Kamlet and Taft. The multiparametric analysis indicates that hydrogen bond donor ability of the solvent (for band I) and non-specific dipolar interactions of the solvents (for band II) play an important role in absorption maxima in pure solvents. Preferential solvation by ethanol (for two bands) was detected in i-PrOH-EtOH solvent mixture. Two spectrophotometric graphical methods were used to estimate the dissociation constant (pK b) with resulting average value of 10.12±0.41.

The solubilities of lanthanum carbonate La2(CO3)3·8H2O in solutionsS 0([H+]=H mol kg−1, [Na+]=(I−H) mol kg−1, [ClO 4 − ]=I mol kg−1) at various fixed partial pressures of CO2 have been investigated at 25.0 °C. The hydrogen ion molality and the total molality of La(III) ion in equilibrium with the solid phase were determined by e.m.f. and analytical methods, respectively. The stoichiometric solubility constants $$\log ^ \star K_{ps0}^I = \log \{ [La^{3 + } ]p_{CO_2 }^{3/2} [H^ + ]^{ - 3} \}$$ according to the overall reaction $$1/2La_2 (CO_3 )_3 \cdot 8H_2 O + 3H^ + \rightleftharpoons La^{3 + } + 3/2CO_2 + 11/2H_2 O$$ have the values 10.23, 10.37, 10.58, 10.77 and 11.06 forI=0.1, 0.25, 1.0, 2.0 and 4.0 mol kg−1 (Na)ClO4, respectively. The extrapolation to infinite dilution using the Pitzer equations resulted in a “thermodynamic” solubility constant: $$\log ^ \star K_{ps0}^0 = \log \{ ([La^{3 + } ]p_{CO_2 }^{3/2} [H^ + ]^{ - 3} )(\gamma _{La^3 } + \gamma _{H^ + }^{ - 3} )a_{H_2 O}^{11/2} \} = 9.57 \pm 0.05$$ . This in turn led to the Gibbs energy of lanthanite, La2(CO3)3·8H2O, formation: $$\Delta _f G_{298}^{O - } = - 5048.4 kJ mol^{ - 1}$$ . Die Löslichkeit von Lanthancarbonat La2(CO3)3·8H2O in LösungenS 0([H+]=H mol kg−1, [Na+]=(I−H) mol kg−1, [ClO 4 − ]=I mol kg−1) wurde bei 25.0 °C und verschiedenen, konstanten Partialdrücken von CO2 untersucht. Die Molalität der Wasserstoff- und die totale Molalität der La(III)-Ionen im Gleichgewicht mit der festen Phase wurden potentiometrisch bzw. analytisch bestimmt. Für die stöchiometrischen Löslichkeitskonstanten $$\log ^ \star K_{ps0}^I = \log \{ [La^{3 + } ]p_{CO_2 }^{3/2} [H^ + ]^{ - 3} \}$$ entsprechend der Gesamtreaktion $$1/2La_2 (CO_3 )_3 \cdot 8H_2 O + 3H^ + \rightleftharpoons La^{3 + } + 3/2CO_2 + 11/2H_2 O$$ wurden beiI=0.1, 0.25, 1.0, 2.0 und 4.0 mol kg−1 (Na)ClO4, folgende Werte gefunden: 10.23, 10.37, 10.58, 10.77 und 11.06. Die Extrapolation auf unendliche Verdünnung mit Hilfe der Pitzer-Gleichungen führte zu einer „thermodynamischen“ Löslichkeitskonstante von: $$\log ^ \star K_{ps0}^0 = \log \{ ([La^{3 + } ]p_{CO_2 }^{3/2} [H^ + ]^{ - 3} )(\gamma _{La^3 } + \gamma _{H^ + }^{ - 3} )a_{H_2 O}^{11/2} \} = 9.57 \pm 0.05$$ . Mit dieser ergab sich die freie Bildungsenthalpie von Lanthanit La2(CO3)3·8H2: zu: $$\Delta _f G_{298}^{O - } = - 5048.4 kJ mol^{ - 1}$$ .

Vitreous coatings can be used as a means for the corrosion protection of metals and alloys due to their excellent dielectric properties. Their use is of particular interest in the case of highly aggressive media (different pH levels, temperatures up to 600 °C). However, ceramic coatings exposed to aggressive agents and thermal cycles can experience devitrification and spalling problems, with the consequent loss of their protective properties.The present study uses the electrochemical impedance spectroscopy technique to characterize, in a non-destructive way, the appearance of surface defects induced by the effect of temperature and chemical attack on a vitreous enamel deposited on steel. By means of analysis of the impedance diagrams obtained, a relationship is established between the loss of the coating's protective properties and the mechanism by which the corrosion process takes place.

We analyzed the role of Ab affinity on Ab-mediated Ag uptake and presentation to T cells. Hen egg white lysozyme (HEL) was captured by bifunctional hybrid proteins (Fv-MalE) in which the variable fragment (Fv) of the anti-HEL mAb D1.3 was covalently linked to the Escherichia coli MalE protein. These complexes were targeted via two anti-MalE mAbs to an APC expressing a receptor for the Ab constant region. The combination of Fv-MalE and anti-MalE mAbs increased, specifically, HEL presentation. With this experimental system, we evaluated the impact of six different mutations, affecting the Fv-MalE complementarity determining regions, on the increase of HEL presentation by the corresponding hybrids. These mutations increase the dissociation rate constant (k(off)), and, thus, the dissociation constant of the HEL/Fv-MalE interaction, up to 650-fold, as compared with the wt Fv-MalE. Increasing the k(off) from 7 x 10(-4) s(-1) up to 300 x 10(-4) s(-1) did not interfere with the enhancement of HEL presentation. A mutant with a k(off) of 600 x 10(-4) s(-1) had a reduced enhancement ability, and mutants with k(off) higher than 5700 x 10(-4) s(-1) did not enhance HEL presentation at all. These results show that affinity determines the efficiency of Ab-mediated Ag presentation to T cells. One consequence is that affinity maturation in specific B lymphocytes can drastically enhance their ability to collaborate with T cells in an MHC-restricted way. This may contribute to the selection of high affinity B cell clones.