Relative Affinity Values Research Articles

Development of novel blend poly (Ethylene Glycol) / Poly(Ethersulfone) polymeric membrane using N-Methyl-2-Pyrollidone and dimethylformamide solvents for facilitating CO2/N2 gas separation

Carbon dioxide (CO2) emissions have been the main cause for anthropogenic climate change. To meet the growing demand for CO2 removal, membrane separation technology using blend membranes is a promising and attractive alternative. Recently, blend membranes consisting of glassy and rubbery polymers has risen in popularity for the gas separation industry due to its ability to effectively minimise trade-off between selectivity and permeability. In this study, glassy poly(ethersulfone) (PES) is blended with rubbery poly(ethylene glycol) (PEG) using a mixture of n-methyl-2-pyrrolidone (NMP) and dimethylformamide (DMF) solvents. Compatibility of PES, PEG, NMP, and DMF within the casting solution was studied using the Hansen solubility parameter (HSP) ternary diagram. Moreover, compatibility of different weight ratio of NMP/DMF solvents (45/15, 40/20, 30/30, 20/40, and 15/45) with respect to the 20/20 PEG/PES polymer blend was investigated based on its relative affinity values. Additionally, single-gas permeation test was used to evaluate the performance of the synthesized PEG/PES blend membranes towards CO2/nitrogen (N2) separation. Scanning electron microscopy, contact angle, and attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) analysis were conducted to analyse the membrane morphology, wettability, and functional groups, respectively. Based on the HSP ternary diagram, PES, PEG, NMP, and DMF were predicted to be miscible in the casting solution. Blends are also deemed more compatible with higher DMF fraction due to the decreasing relative affinity values with increasing DMF fraction. According to the results, NMP/DMF solvent concentration was optimized at 20/40 NMP/DMF having obtained the highest CO2/N2 selectivity of 1.36 ± 0.01 and CO2 permeance of 6277.45 ± 11.0 GPU. On that regard, the enhanced CO2/N2 gas separation performance of the PEG/PES blend membrane can potentially have an important contribution to CO2 separation processes in the industry in order to decrease emissions and minimize the risk of climate change.

Binding of Human Nucleotide Exchange Factors to Heat Shock Protein 70 (Hsp70) Generates Functionally Distinct Complexes in Vitro

Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone heat shock protein 70 (Hsp70). There are six BAG family NEFs in humans, and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete for binding to Hsp70 or how they might differentially shape its biochemical activities. Toward these questions, we measured the binding of human Hsp72 (HSPA1A) to BAG1, BAG2, BAG3, and the unrelated NEF Hsp105. These studies revealed a clear hierarchy of affinities: BAG3 > BAG1 > Hsp105 ≫ BAG2. All of the NEFs competed for binding to Hsp70, and their relative affinity values predicted their potency in nucleotide and peptide release assays. Finally, we combined the Hsp70-NEF pairs with cochaperones of the J protein family (DnaJA1, DnaJA2, DnaJB1, and DnaJB4) to generate 16 permutations. The activity of the combinations in ATPase and luciferase refolding assays were dependent on the identity and stoichiometry of both the J protein and NEF so that some combinations were potent chaperones, whereas others were inactive. Given the number and diversity of cochaperones in mammals, it is likely that combinatorial assembly could generate a large number of distinct permutations.

Interferon-β antibodies have a higher affinity in patients with neutralizing antibodies compared to patients with non-neutralizing antibodies

In this study, we investigated the affinity, determined by a relative affinity assay, using increasing concentrations of sodium-isothiocyanate to disrupt the antigen antibody binding of neutralizing and non-neutralizing antibodies against interferon-β (IFNβ)-1a and -1b in 73 serum samples of MS patients treated with IFNβ-1a or -1b. Relative affinity values were significantly higher in NAB-positive compared to NAB-negative samples and in samples of IFNβ-1a-treated patients compared to IFNβ-1b. A significant positive correlation between relative affinity values and therapy duration indicates affinity maturation as another qualitative factor in IFNβ neutralization.

Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells: REGULATION OF RECEPTOR DOWN-REGULATION BY HETERODIMERIZATION

Although previous pharmacological and biochemical data support the notion that muscarinic acetylcholine receptors (mAChR) form homo- and heterodimers, the existence of mAChR oligomers in live cells is still a matter of controversy. Here we used bioluminescence resonance energy transfer to demonstrate that M1, M2, and M3 mAChR can form constitutive homo- and heterodimers in living HEK 293 cells. Quantitative bioluminescence resonance energy transfer analysis has revealed that the cell receptor population in cells expressing a single subtype of M1, M2, or M3 mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M1/M2, M2/M3, and M1/M3 mAChR heterodimers, although the relative affinity values were slightly lower than those for mAChR homodimers. Short term agonist treatment did not modify the oligomeric status of homo- and heterodimers. When expressed in JEG-3 cells, the M2 receptor exhibits much higher susceptibility than the M3 receptor to agonist-induced down-regulation. Coexpression of M3 mAChR with increasing amounts of the M2 subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M3, suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.

Distribution of Aged 14C−PCB and 14C−PAH Residues in Particle-Size and Humic Fractions of an Agricultural Soil

Organic matter is considered to be the single most importantfactor limiting availability and mobility of persistent organic pollutants (POPs) in soil. This study aimed to characterize the distribution of 14C-PCB (congeners 28 and 52) and 14C-PAH (fluoranthene and benzo[a]pyrene) residues in an Orthic Luvisol soil obtained from two lysimeter studies initiated in 1990 at the Agrosphere Institute (Forschungszentrum Jülich GmbH, Germany). The lysimeter soils contained a low-density OM fraction, isolated during soil washing, which contained a significant fraction (3-12%) of the total 14C-activity. Soils were also fractionated according to three particle sizes: >20, 20-2, and <2 microm. Relative affinity values of 14C-activity for the different particle sizes varied in the order 20-2 microm > (<2 microm) approximately (>20 microm) for the PCBs. Relative affinity values of 14C-activity for the different particle sizes varied in the order 20-2 microm > (<2 microm) > (>20 microm) for the PAHs. The distribution of 14C-PCB or 14C-PAH residues in the organic and inorganic matrixes of the particle-size fractions was determined using methyl isobutyl ketone (MIBK). 14C-PCB and 14C-PAH-associated activities were primarily located in the humin fraction of the 20-2 and <2 microm particle-size fractions of the soil. A small fraction was associated with the fulvic and humic acid fractions; these were quantitatively more important for the PAHs than the PCBs. There appeared to be a high degree of association of 14C-activity with the mineral fraction following MIBK separation of the humic fractions, ranging between 8 and 52% for 14C-PCBs and 57-80% for 14C-PAHs. The mineral (inorganic) component of the soils apparently played a significant (previously unreported) role in the sequestration of both PCBs 28 and 52 and the PAHs fluoranthene and benzo[a]pyrene.

Optimization of tether length in nonglycosidically linked bivalent ligands that target sites 2 and 1 of a Shiga-like toxin.

A series of bivalent ligands for a Shiga-like toxin have been synthesized, their experimentally determined inhibitory activities were compared with a simplified thermodynamic model, and computer simulations were used to predict the optimal tether length in bivalent ligands. The design of the inhibitors exploits the proximity of the C-2' hydroxyl groups of two P(k)-trisaccharides when bound to two different, neighboring carbohydrate recognizing binding sites located on the surface of Shiga-like toxin. NMR studies of the complex between the toxin and bivalent ligands show that site 2 and site 1 of a single B subunit are simultaneously occupied by a tethered P(k)-trisaccharide dimer. A simplified thermodynamic treatment provides the intrinsic affinities and binding energies for the intermolecular and intramolecular association events and permits the deconvolution of the contributions to the relative binding energies for the set of bivalent ligands. Conformational analysis based on MD simulations for bivalent galabioside dimers containing different tethers demonstrated that the calculated local concentrations of the pendant ligand at the second binding site correlate with the experimentally determined relative affinity values of the respective bivalent ligands, thereby providing a predictive method to optimize tether length.

Improvement in the Properties of 3‐Phenyl‐3‐trifluoromethyldiazirine Based Photoreactive Bis‐glucose Probes for GLUT4 Following Substitution on the Phenyl Ring.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Improvement in the properties of 3-phenyl-3-trifluoromethyldiazirine based photoreactive bis-glucose probes for GLUT4 following substitution on the phenyl ring.

We have developed two novel 3-phenyl-3-trifluoromethyldiazirinyl bis-glucose derivatives to investigate the properties of the adipocyte glucose transporter GLUT4. These compounds were substituted by electron-withdrawing (iodo and nitro) groups on the aromatic ring of 3-phenyl-3-trifluoromethyldiazirine photophore and were found to be more photosensitive than compounds without such substituents. The compounds were used as inhibitors of insulin-stimulated glucose transport activity in order to assess half-maximal inhibition or relative affinity values for GLUT4. The affinities were found to be 60-130 times higher than the parent compound D-glucose. Because of the increased photo-reactivity and high affinity these compounds will be useful in studies directed at further elucidation of GLUT4 function.

Phage Display Cloning and Characterization of Monoclonal Antibody Genes and Recombinant Fab Fragment against the CD98 Oncoprotein

The Fab gene of anti‐CD98 heavy chain (h.c.) monoclonal antibody (mAb) HBJ127 was cloned and expressed as a recombinant Fab (rFab) fragment by means of a phage display system. The variable heavy and light chain genes of HBJ127 were found to be derived from VOx‐1 and IgVk8‐30 germline, respectively. Extensive somatic mutation was found in the heavy chain complementarity determining region 2. rFab fragment was purified homogeneously from crude bacterial lysates by Ni‐chelate chromatography in a yield of 71.4 μg from 100 ml of culture. rFab fragment was reactive with the cell surface of CD98‐positive cells irrespective of tissues of origin, but not with CD98‐neg‐ative cells. The recognition site of the rFab fragment was identical to that of mAb since the binding of rFab fragment to HeLaS3cells was completely inhibited by pretreatment with an excess of mAb. The relative affinity values of rFab fragment and mAb were found to be 0.11x108 and 0.35X108M‐1, respectively. Three‐fold lower affinity of rFab fragment may be due to the difference of valency of the antibody preparation. Cell growth inhibition in vitro by rFab fragment preincubated with anti‐Fab suggests that the rFab fragment produced by cloned gene‐bearing Escherichia coli was identical to the Fab part of HBJ127 mAb. These results show that a small fragment with antigen binding activity similar to that of the parent mAb can easily be prepared by using a phage display system. To our knowledge, this is a first report of the production of anti‐CD98 h.c. rFab fragment.

In vitro scanning saturation mutagenesis of an antibody binding pocket.

We have combined PCR mutagenesis with in vitro transcription/translation and ELISA for the rapid generation and characterization of antibody mutants. The PCR products are used directly as the template for the in vitro transcription/translation reactions and because no cloning steps are required, the in vitro saturation mutagenesis of one residue can be completed in duplicate within a week by a single investigator. In vitro scanning saturation mutagenesis was used to analyze the role and plasticity of six key contact residues (H:Tyr-33, H:Asn-35, H:Tyr-50, H:Trp-100, L:Val-94, and L:Pro-96) in the binding pocket of a single chain Fv antibody derived from the 26-10 monoclonal antibody. A total of 114 mutant antibodies were produced; all 19 substitutions at each of the 6 chosen positions. The mutants were analyzed for binding to digoxin, digitoxin, digoxigenin, and ouabain resulting in the generation of a comprehensive data base of 456 relative affinity values. Excellent agreement between the relative affinity values obtained with in vitro synthesized mutant antibodies and equilibrium affinity data obtained with previously reported purified mutant monoclonal antibodies was observed. Approximately 75% of the single amino acid mutants exhibited significant binding to one or more of the digoxin analogs. Mutations that alter and, in some cases, reverse specificity for the different digoxin analogs were identified. In vitro scanning saturation mutagenesis represents a new tool for protein structure-function and engineering studies and can be interfaced with laboratory automation so that an even higher throughput of protein mutants can be constructed and analyzed.

New methods for determining dissociation constants of agonist-receptor complexes

Methods are presented which enables the dissociation constant of agonist-receptor complexes (KA) to be estimated without knowledge of the relative intrinsic efficacies. The dissociation constants are estimated from fixed agonist-competitive antagonist concentration combinations which simulate partial agonists. In general, estimating of KA by employing these methods require ф and KB to be known; where ф = antagonist]/[agonist] and KB is the dissociation constant of the competitive antagonist-receptor complex. Some of these methods require only knowledge of ф, while KB may be unknown. A method is also described to estimate KA without knowing the value of KB and the competitive antagonist concentration. In addition to the estimation of KA values, a new method for estimation of the dissociation constants of competitive antagonist-receptor complexes (KB) is reported. One of the new methods was exemplified practically by using sets of experimental agonist concentration-effect curves determined in the absence and presence of increasing concentrations of competitive antagonists. From this pharmacological data the apparent KA value for carbachol (in muscarinic M3 receptors of the guinea-pig ileum) was determined. This action illustrated that the apparent affinity values (KA) and height of the stimulus curve of an agonist may be determined from published pharmacological data. This procedure affords a possibility to establish whether or not spare receptors are present in a particular system. It was also shown that relative affinity values provide more reliable information than does isolated KA values.

Site‐directed mutagenesis and epitope‐mapped monoclonal antibodies define a catalytically important conformational difference between human placental and germ cell alkaline phosphatase

Placental (PLAP) and germ cell (GCAP) alkaline phosphatases were probed immunologically with a library of 18 murine monoclonal antibodies reacting with different conformational epitopes on PLAP. Three main antigenic domains (I, II and III) were mapped by antibody competition experiments and the relative binding of the antibodies to site-directed PLAP mutants. Relative affinities of each of the antibodies for the wild type (wt) GCAP were 2-3-fold lower than the values found for wt PLAP. Relative affinity was determined for a series of PLAP mutants, in which one, two or three amino acids were substituted for the corresponding wt GCAP residues by site-directed mutagenesis. Substitutions at residues 15, 38, 67, 241 or 254 induced a major decrease in affinity (6-10-fold) primarily for those antibodies reacting within domain I, whereas changes at positions 84 and 297 led to a 2-3-fold enhancement of affinities as measured with antibodies reacting within the three domains. Arg209 was found to constitute the only difference between the S and F allelic phenotypes of PLAP and to structure the epitope for the F/S allotype-discriminating antibodies. Arg241 was found to constitute the epitope for the antibody 17E3 that discriminates between PLAP and GCAP. Mutagenesis at position 68 or 133 had little effect on the overall reactivity with the antibody panel. Substitution in wt PLAP of Glu429 for Gly429 or even for His429 (found at this position in tissue-nonspecific alkaline phosphatase) and Ser429 (found in the intestinal alkaline phosphatase) induced a general decrease in affinities as detected by 16 of the 18 antibodies. The conformational change accompanying mutagenesis of Glu429 in PLAP, is important in view of the recent identification of Gly429 as the major determinant of the unique GCAP inhibition by the uncompetitive inhibitor L-Leu. Relative affinity values determined for the rare L-Leu sensitive heterodimeric FD and SD PLAP phenotypes, suggested that the reactivity pattern of the D homodimer with the antibody panel, would resemble more closely that of wt GCAP than wt PLAP. Our data suggest that the uncompetitive inhibition of GCAP by L-Leu is due to an enzymatically critical conformational change in a loop region proximal to the active site of the enzyme, induced by substitution of a single amino acid residue.

The modeled structure of the IgG Gar VL region and its implications for anti-flavin and anti-DNP fine specificities.

The variable domain (V) sequence of the lambda-light chain of the Gar IgG2 human myeloma protein was determined from enzymatically generated peptides that were isolated, characterized, and ordered by overlap and/or by subgroup homology. The sequence consisted of the amino terminal 107 residues of the light chain, and shared subgroup V lambda III-specific residues as well as a pattern of CDR deletions unique to most V lambda III sequences. The structural role of the light V region in the high affinity for flavins and the low affinity for 2,4-dinitrophenyl haptens characteristic of the intact IgG molecule was coarsely assessed by way of model building and by comparison with the refined combining site model of MOPC 315, an Ig with relative affinity values for DNP and riboflavin the converse of those observed in Gar.

External diffusion effects on the kinetic constants of immobilized enzyme systems

In order to further our understanding of immobilized enzyme reaction kinetics, the effects of external diffusion on the kinetic constants are studied for various reaction systems. It is shown that the variations of apparent kinetic constants with diffusional limitations are not the same as those of Michaelis-Menten kinetics, although both the inhibition kinetics and the two substrate reaction kinetics can be expressed in the form of simple Michaelis-Menten type by using apparent rate parameters. We found that there will be changes in the apparent kinetic constants depending not only on the types of enzyme reaction kinetics but also on the relative rate of diffusion or flow-through in the microenvironment. For substrate inhibition kinetics, both the apparent maximum reaction rate and the apparent Michaelis constant decrease while the apparent maximum reaction rate for product inhibition kinetics is increased and the apparent Michaelis constant decrease as mass transfer limitation is reduced. In the case of two substrate enzyme reaction kinetics, as the diffusional limitation is reduced the apparent maximum reaction rate increase but the apparent Michaelis constant can increase, decrease or remain nearly constant depending on the values of relative affinity and on the fixed substrate concentrations. The results of theoretical analyses are compared with the experimental data obtained and reported previously, and a very good agreement was found.

The binding of tamoxifen to oestrogen receptor proteins under equilibrium and non-equilibrium conditions

Data have been presented from sucrose density gradient analysis, protamine sulphate precipitation and specificity studies to show that oestradiol-17β and tamoxifen share a common binding protein. In addition, the binding of [ 3H ]-tamoxifen to cytosol preparations from human and rat mammary adenocarcinomas and rat uteri was measured under equilibrium conditions using Scatchard analysis. The relative affinity values obtained were, however, greater than those calculated from competitive binding studies. In addition the competition between tamoxifen and oestradiol- 17β for oestrogen binding sites at 4°C decreased with increasing incubation time. Based on competitive binding experiments, the principal metabolite of tamoxifen, metabolite B, associated with oestrogen receptor proteins with a higher affinity than tamoxifen. Kinetic studies indicate that these variations probably result from the relative dissociation rates of metabolite B and tamoxifen from the oestrogen receptor, tamoxifen dissociating much more rapidly than metabolite B. It is also proposed that kinetic studies may have important implications with respect to the mechanism of action of tamoxifen.