Observed Kd Values Research Articles

A Simple One-Parameter Percent Dissolved Versus Time Dissolution Equation that Accommodates Sink and Non-sink Conditions via Drug Solubility and Dissolution Volume

In vitro dissolution generally involves sink conditions, so dissolution equations generally do not need to accommodate non-sink conditions. Greater use of biorelevant media, which are typically less able to provide sink conditions than pharmaceutical surfactants, necessitates equations that accommodate non-sink conditions. One objective was to derive an integrated, one-parameter dissolution equation for percent dissolved versus time that accommodates non-sink effects via drug solubility and dissolution volume parameters, including incomplete solubility. A second objective was to characterize the novel equation by fitting it to biorelevant dissolution profiles of tablets of two poorly water-soluble drugs, as well as by conducting simulations of the effect of dose on dissolution profile. The Polli dissolution equation was derived, % ;dissolved=100%left[1-frac{left({{M}_{0}-c}_{s}Vright)}{{M}_{0}-{{c}_{s}Ve}^{{-k}_{d}left(frac{{{M}_{0}-c}_{s}V}{V}right)t}}right], where M0 is the drug dose (mg), cs is drug solubility (mg/ml), V is dissolution volume (ml), and kd is dissolution rate coefficient (ml/mg per min). Maximum allowable percent dissolved was determined by drug solubility and not a fitted extent of dissolution parameter. The equation fit tablet profiles in the presence and absence of sink conditions, using a single fitted parameter, kd, and where solubility ranged over a 1000-fold range. kd was generally smaller when cs was larger. FeSSGF provided relatively small kd values, reflecting FeSSGF colloids are large and slowly diffusing. Simulations showed impact of non-sink conditions, as well as plausible kd values for various cs scenarios, in agreement with observed kd values. The equation has advantages over first-order and z-factor dissolution rate equations. An Excel file for regression is provided.Graphical

Variability in 210Pb and 210Po partition coefficients (Kd) along the US GEOTRACES Arctic transect

The naturally occurring 210Po (t1/2 = 138.3d) and 210Pb (t1/2 = 22.3 y) radionuclide pair has been extensively used to study particle scavenging, cycling and transport in the ocean. However, the role of particle composition in the scavenging, sorption and fractionation of 210Po210Pb needs to be better understood to successfully utilize these radionuclides as oceanographic tracers. Here, we measure dissolved and particulate 210Pb and 210Po activities for both small (1-51 μm) and large particles (> 51 μm) with abundance of major phases of suspended particulate matter (SPM) from the US GEOTRACES Arctic transect to examine the role of the concentration and composition of SPM in scavenging and fractionation of these radionuclides along the coastal, Canada Basin and interior stations. 210Po210Pb disequilibria were observed throughout the water column for all stations. The Pacific end member had the highest activity of 210Po and 210Pb in dissolved samples. The calculated partition coefficients (Kd) of 210Po and 210Pb varied between 3.2 × 105 to 9.9 × 108 L kg−1 and 7.9 × 105 to 6.9 × 108 L kg−1 respectively and followed a log-linear trend with SPM for particle size fractions. Principal Component Analysis (PCA) and correlation analysis for six major particle phases indicated a significant positive correlation between 210Pb and the lithogenic phase. Both 210Po and 210Pb had significant negative correlations with opal. Our results suggest that particle composition alone cannot explain the observed variability in Kd across the different oceanic regime along the transect. Estimated Kd values from an end member mixing model were found to be within the range of observed Kd values and suggest that higher particulate Mn concentration in the Arctic may be partly responsible for the higher Kd observed in the basin compared to Atlantic and Pacific.

Calcium isotope separation by band chromatography using 18-crown-6-ether resin

ABSTRACT To develop the 48Ca enrichment process, a feasibility study on a band chromatography was made using 9 M HCl solution and crown ether resin synthesized in porous silica beads. Prior to the chromatographic experiments, distribution coefficients, Kd, of Ca2+ and Sr2+ were measured at different concentrations of these ionic species. The frontal boundary of the chromatography was made by a usual manner of the breakthrough mode of calcium feeding, and the rear boundary was made by introducing strontium as a following ion on the basis of the observed Kd values. It was confirmed that the heavy isotope 48Ca was depleted in the rear boundary region, while 48Ca was enriched in the front boundary region. The values of separation coefficient ε (= α – 1) in three chromatographic operations at different temperatures were observed as 2 × 10−3 ~ 3 × 10−3. The separation coefficients observed in the front boundary regions, where 48Ca was enriched, agreed with those observed in the rear boundary regions, where 40Ca was enriched.

An analytical method for detecting toxic metal cations using cyclotriveratrylene derivative capped gold nanoparticles

Cyclotriveratrylene-oxime (CTV-oxime) derivatives that terminate with a dithiolate linker were synthesized enabling the supramolecular scaffold to adhere to gold nanoparticles (AuNPs) with the bowl-shaped cavity of the CTV scaffold exposed for utilization in host–guest chemistry. Exposure of these CTV functionalized AuNPs to varying concentrations of di- and trivalent metal cations resulted in the formation of large CTV-AuNP polymeric clusters and an accompanying a shift in the plasmon resonance. These interactions between the CTV-AuNPs and the metal cations in solution provides proof-of-concept that supramolecular functionalized AuNPs can be used as a simple and straightforward, on-site detection system for toxic metal cations in solution. The order of binding affinity of the metals studied based on observed Kd values is Cu2+>Zn2+>Pb2+>Hg2+>Eu3+>Cd2+.

Soil factors affecting solubility and mobility of zinc in contaminated soils

In view of modern worldwide research carried out in the context of broadly understood soil chemistry and monitoring of the natural environment, the methods of assessing the risk of Zn contamination in soil and agricultural crops based on the analyses of Zn concentration in the soil solution have gained more recognition. Accordingly, research on the evaluation of the effects of selected soil properties on changes in solid/solution partitioning coefficient (K d), the total concentration and activity of zinc in the soil solution was undertaken in this study. The study was based on the microplot field experiment. Investigated soil factors were as follows: soil texture, pH, organic carbon content and the degree of Zn contamination. The results indicated that zinc activity in the soil solutions was very high and comparable to the total concentration of zinc. The investigated soil properties significantly influenced the K d, the total concentration of zinc, as well as the concentration of Zn2+ in the soil solution. The total concentration and activity of zinc in the soil solution increased with increasing Zn content in the soil and rising soil acidity, while they decreased with increasing the content of organic carbon and clay particles. The values of K d increased significantly with rising soil pH and total Zn content and decreased with increasing soil organic content. Observed K d values were significantly higher in soil with 13 % clay than in soil with 7 % clay.

Molecular Mechanism of SSR128129E, an Extracellularly Acting, Small-Molecule, Allosteric Inhibitor of FGF Receptor Signaling

The fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling network plays an important role in cell growth, survival, differentiation, and angiogenesis. Deregulation of FGFR signaling can lead to cancer development. Here, we report an FGFR inhibitor, SSR128129E (SSR), that binds to the extracellular part of the receptor. SSR does not compete with FGF for binding to FGFR but inhibits FGF-induced signaling linked to FGFR internalization in an allosteric manner, as shown by crystallography studies, nuclear magnetic resonance, Fourier transform infrared spectroscopy, molecular dynamics simulations, free energy calculations, structure-activity relationship analysis, and FGFR mutagenesis. Overall, SSR is a small molecule allosteric inhibitor of FGF/FGFR signaling, acting via binding to the extracellular part of the FGFR.

Phase partitioning, retention kinetics, and leaching of fumigant methyl iodide in agricultural soils

Although it is not currently being sold in the USA, the recent US registration of the fumigant methyl iodide has led to an increased interest in its environmental fate and transport. Although some work has now considered its volatile emissions from soil, there remains a lack of experimental data regarding its ability to be retained in soil and ultimately become transported with irrigation/rain waters. Using laboratory batch and soil column experiments, we aimed to better understand the phase partitioning of MeI, the ability of soils to retain MeI on the solid phase, and the potential for leaching of MeI and its primary degradation product, iodide, down a soil profile. Results indicated that MeI was retained by the solid phase of soil, being protected from volatilization and degradation, particularly in the presence of elevated organic matter. Retention was greater at lower moisture content, and maximum retention occurred after 56days of incubation. At higher moisture content, the liquid phase also became important in retaining MeI within soil. Together with low observed KD values (0.10 to 0.57mLg−1), these data suggest that MeI may be prone to leaching. Indeed, in a steady-state soil column study, initially retained MeI was transported with interstitial water. The MeI degradation product, iodide, was also readily transported in this manner. The data highlight a potentially significant process by which MeI fate and transport within the environment may be impacted.

農耕地土壌における放射性ニッケルの土壌‐土壌溶液分配係数 統計的処理による分配係数推定の試み

Soil-soil solution distribution coefficient (Kd) is an important parameter in radiation dose assessment models for the environmental transfer of radionuclides. Since Kd values vary with soil properties, development of their estimation method using some soil properties is useful. In this study, we estimated Kd values of radionickel using selected soil properties. We determined Kd values of Ni for 142 agricultural soil samples collected throughout Japan and measured such soil properties as pH, cation exchangeable capacity, exchangeable Ca, clay content, concentrations of major elements in soil, and concentrations of major elements in water soluble fractions. The relationship between the Kd of Ni and each soil property was calculated using Spearman's rank correlation test. Then, in order to estimate Kd values of Ni based on soil properties, we used the method which we developed in the previous study for strontium using multiple linear regression analysis. Our results showed that potassium concentration in soil and phosphorus concentration in water soluble fraction were the most important factors contributing to estimation of Kd values. Finally, a predictive expression including three selected variables was created to estimate Kd values of Ni. Predicted Kd values from the expression were in good agreement with observed Kd values.

Is Dimerization Required for the Catalytic Activity of Bacterial Biotin Carboxylase?

Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. The biotin carboxylase (BC) subunit of Escherichia coli ACC is believed to be active only as a dimer, although the crystal structure shows that the active site of each monomer is 25 A from the dimer interface. We report here biochemical, biophysical, and structural characterizations of BC carrying single-site mutations in the dimer interface. Our studies demonstrate that two of the mutants, R19E and E23R, are monomeric in solution but have only a 3-fold loss in catalytic activity. The crystal structures of the E23R and F363A mutants show that they can still form the correct dimer at high concentrations. Our data suggest that dimerization is not an absolute requirement for the catalytic activity of the E. coli BC subunit, and we propose a new model for the molecular mechanism of action for BC in multisubunit and multidomain ACCs.

Arsenite binding to synthetic peptides: The effect of increasing length between two cysteines

We utilized radioactive 73As-labeled arsenite and vacuum filtration methodology to determine the binding affinity of arsenite to eight synthetic peptides ranging from 13 to 24 amino acids long and containing one or two cysteines separated by 0-17 intervening amino acids. Six of the eight peptides were highly similar in amino acid sequence and were based on cysteine containing regions of the hormone-binding site of the human estrogen receptor-alpha (e.g., the sequence of peptide 28 is LEGAWCGKGVEGTEHLYSMKCKNV). The peptides with 0-14 intervening amino acids between two cysteines bound arsenite with Kd values of 2.7-20.1 uM and with Bmax values from 36 to 103 nmol/mg protein (from 0.083 to 0.19 nmol/nmol of protein). Thus, increasing the number of intervening amino acids from 0 to 14 made very little difference in the observed Kd values for arsenite, a surprising finding. Therefore, these peptides are flexible in solution and effectively contain a dithiol high affinity binding site for arsenite. Peptide 17 with two C separated by 19 amino acids bound arsenite with a Kd of 123 uM and a Bmax of 41.8 nmol/mg. The monothiol peptide 19 bound arsenite with a Kd of 124 uM and a Bmax of 26 nmol/mg protein. All experimental binding curves fit well to a one site binding model.

Multiple Activation States of Integrin α4β1 Detected through Their Different Affinities for a Small Molecule Ligand

We have used the highly specific alpha4beta1 inhibitor 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study alpha4beta1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha4beta1. Observed KD values for BIO1211 binding ranged from a value of 20-40 nM in the non-activated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to 100 pM in the activated state seen in 2 mM Mn2+ to 18 pM when binding was measured after co-activation by 2 mM Mn2+ plus 10 microgram/ml of the integrin-activating monoclonal antibody TS2/16. The large range in KD values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 x 10(-4) s-1 to >140 x 10(-4) s-1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 x 10(6) M-1 s-1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on alpha4beta1 that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to alpha4beta1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.

Chimeric Rat/Human Neurotensin Receptors Localize a Region of the Receptor Sensitive to Binding of a Novel, Species-specific, Picomolar Affinity Peptide

Recently, we reported the development of a species-specific neurotensin analog that displays selective binding affinity at the rat and human neurotensin (NT) receptor, L-[3,2'-Nal11]NT(8-13) (where Nal is naphthylalanine) (NT19). We have developed another neurotensin analog, L-[3,1'-Nal11]NT(8-13), (NT34), that exhibits a 126-fold difference in binding affinities between the rat and human receptors. This compound differs from our previous reported species-specific ligand in the steric positioning of the naphthyl ring on the L-alanine side chain. For NT34, the observed Kd values at the rat and human neurotensin receptors were 0.046 and 5.8 nM, respectively. In stimulating phosphatidylinositol turnover, the observed EC50 values were 2.8 nM and 130 nM in rat and human, respectively. We constructed a series of chimeric rat/human neurotensin receptor genes and expressed them by transient transfection into human embryonic kidney (HEK-293) cells. Radioligand binding assays were then performed using neurotensin and NT34. Our results led us to propose a region of the neurotensin receptor that may be involved in determining species specificity, i. e. the transmembrane VI, the third extracellular loop, and transmembrane VII regions of the neurotensin receptor.

Up-Regulation of Endothelin-B Receptors in Atherosclerotic Human Coronary Arteries

Both endothelin-A (ETA) and endothelin-B (ETB) receptors are known to be present in human coronary arteries. However, their absolute and relative amounts, functional roles, and the influence of pathology are uncertain. The goal of the present study was to characterize endothelin receptors mediating constriction in human coronary arteries and to assess the influence of cardiomyopathy (CMP) and coronary artery disease (CAD) on ET receptors in human tissue. For comparison, porcine coronary arteries were evaluated in parallel. Competition binding experiments using [125I]ET-1 and different selective and nonselective ETA- and ETB-receptor agonists or antagonists revealed similar relative densities (relative Bmax) of ETA and ETB receptors in coronary arteries from human cardiomyopathic hearts (83% ETA and 17% ETB; n = 5) and porcine hearts (78% ETA and 22% ETB; n = 5). In marked contrast, the relative Bmax of ETB receptors were significantly higher in coronary arteries from human atherosclerotic hearts (51% ETA and 49% ETB; n = 3). Total receptor density (Bmax; fmol/mg protein) was highest in porcine (385 +/- 29) arteries, followed by human CAD (253 +/- 41) and CMP (174 +/- 20) coronary arteries. The relative and absolute Bmax values for ETA and ETB receptors in coronary arteries from a donor heart were similar to those obtained in CMP hearts. There were no significant differences in affinity constants (KD) values for ET-1, ET-3, Sarafotoxin S6c (SRTX S6c), BQ-123, and bosentan (Ro 47-0203) between tissues. In human coronary arteries from CMP hearts, ET-induced constriction seemed to be solely mediated via ETA receptors. In contrast, in porcine coronary arteries 20% of the maximal effect mediated by ET-1 could be attributed to ETB receptors, in agreement with the binding data. The functional role of ETB receptors in CAD tissue could not be evaluated because of the occurrence of spontaneous phasic contractions. We conclude that ETB receptors are up-regulated in human atherosclerotic coronary arteries. Further studies are needed to determine the pathophysiological importance of these receptors.

Diabetes and proteolysis: effects on carnitine palmitoyltransferase-I and malonyl-CoA binding.

Malonyl-CoA binding and malonyl-CoA inhibition of carnitine palmitoyltransferase-I (CPT-I) were measured in hepatic mitochondria from normal and diabetic rats and in protease-treated mitochondria from fed rats to test the hypothesis that proteolysis represents a mechanism by which diabetes produces changes in the sensitivity of CPT-I to inhibition by malonyl-CoA. As in diabetes, protease treatment increased the apparent Ki values for malonyl-CoA. Palmitoyl-CoA greatly diminished malonyl-CoA specific binding in the mitochondrial system being studied, suggesting strong competition at the malonyl-CoA binding site. Proteolysis decreased capacity for specific binding of malonyl-CoA by 60-80%, but it had no effect on binding affinity. In contrast, the decreased specific binding of malonyl-CoA seen in the diabetic state is accompanied by increased binding affinity. Furthermore, observed Kd values differed from Ki values by a factor of 10 or more, suggesting that measured Kd and Ki may represent different ligand-protein complexes. These data suggest that alterations in inhibition of CPT-I by malonyl-CoA occurring in the diabetic state may involve mechanisms other than simple proteolytic removal of malonyl-CoA binding sites.

Synthesis and properties of a potential extracellular fluorescent probe for potassium

Development of a new, selective, high affinity, fluorescent probe CD222, 2, for potassium is described. Coincidentally two related probes, CDF18, 3, and CTF18, 4, were prepared and showed selectivity for the cations sodium and calcium, respectively, and possible reasons for these differences are discussed. The bicyclic probe CD222, 2, with observed KD values for K+ ranging from 1 to 10 mmol dm–3, has potential applications as an extracellular probe for potassium.

Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. Identification of sites of component interaction.

Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporium OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (alpha beta gamma)2) is shown to significantly change the electronic environment of the mu-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II).Fe(III)) and fully reduced (Fe(II).Fe(II)) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase have been shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (KD approximately 0.4 microM) or hydroxylase (two binding sites, KDa approximately 10 nM, KDb approximately 8 microM). The observed KD values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase alpha and beta subunits, respectively. The alpha and beta subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occurred. This effect was inhibited by the presence of hydroxylase and was accompanied by a loss of the ability of the component B to bind to the hydroxylase. Thus, formation of a component B-hydroxylase complex is apparently required for effective catalysis linked to NADH oxidation. When present in concentrations greater than required to saturate the initial hydroxylase complex, component B inhibited both the rate of the enzymic reaction and the cross-linking of the reductase to the hydroxylase. This suggests that a second complex involving component B can form that negatively regulates catalysis by preventing formation of the reductase-hydroxylase complex.

Ion exchange separation of Eu(III), Th(IV), U(VI) and Pu(IV) ions on Amberlyst A-15 in non-aqueous solutions

Distribution ratios of europium(III), thorium(IV), uranium(VI) and plutonium(IV) ions on Amberlyst A-15, a macroreticular polystyrene sulfonate resin, after extraction in HTTA-TBP-Shell Sol-T and HTTA-TOPO-benzene solutions have been determined as a function of the aqueous acidity. The affinity orders were Eu≫Pu>Th>U and Eu>Th>Pu>U in the former and the latter solutions, respectively. Separation factors were computed from the observed Kd values. A procedure for the separation of a mixture of Eu(III), Th(IV), and U(VI) ions in HTTA-TOPO-benzene solution in an ion-exchange column is described.

Kinetic properties of the accumulation of 3H-raclopride in the mouse brain in vivo.

The kinetic properties of the accumulation of 3H-raclopride, a selective dopamine (DA) D-2 receptor antagonist, in mouse striatum in vivo was examined under various experimental conditions. The accumulation in striatum was saturable in contrast to that in cerebellum, which linearily increased with the dose. The specific binding of 3H-raclopride in the striatum, defined as the difference in the accumulation in striatum and cerebellum 30 min after the injection was completely inhibited by the D-2 receptor antagonists spiperone and (+)-butaclamol [but not (-)-butaclamol] and the DA receptor agonist N-n-propylnorapomorphine. The mean Bmax value of the specific binding was 40.7 +/- 2.8 pmol/g tissue and the mean apparent KD value, based on the dose injected, was 87.8 +/- 11.5 nmol/kg i.v. (18 different experiments). Pretreatment of the mice with a single injection of reserpine 4 h or 3 days beforehand reduced the apparent KD value which in part seemed to be due to the decreased concentration of synaptic DA. Similarly, gamma-butyrolactone injected immediately before raclopride reduced the apparent KD value, whereas amfonelic acid and (-)-amphetamine increased the observed KD values. These findings indicate competition between endogenous DA and raclopride for the D-2 receptors. Both reserpine and gamma-butyrolactone increased the apparent Bmax value by about 50% which indicates a receptor pool of DA for which raclopride does not compete.

Ion exchange studies of U(VI), Th(IV), Pu(IV) and Eu(III) on a macroreticular resin in TBP-Shell Sol-T solutions

Ion exchange studies of uranium(VI), thorium(IV), plutonium(IV) and europium(III) ions on a macroreticular cation exchange resin, Amberlyst A-15, from solutions of 30% and 5% TBP—Shell Sol-T have been carried out. The metal ions were extracted into TBP Shell Sol-T phase from 8M NH4NO3 at different nitric acid concentrations. Ion exchange distribution ratios as a function of organic phase acidity of 30% and 5% TBP have been computed. Separation factors computed from the observed Kd values are plotted as a function of organic phase acidity.

Ion-sieve properties of an inorganic gel loaded with some complex cations of cobalt(III) a radiochemical study

Distribution coefficients of a number of uni-, di-, tri- and quadrivalent metal ions have been determined on chromium ferrocyanide gel loaded with [Co(NH3)6]3+ and [Co(en)3]3+ ions at 25 °C, and the observed Kd values are compared with those obtained on the unloaded exchanger in H-form. The gel is found to develop ion-sieve properties after being loaded with the complex cations. The variation in Kd values with the pH of Rb+, Cs+, Ti+ and Ag+ solutions has also been studied to understand the mechanism of their uptake.