Total Binding Sites Research Articles

The Self-Assembly of Cationic Metal Complexes on Gold Nanoparticle Surface.

This work aims to study the interaction between cationic metal complexes (M z +) and gold nanoparticles (AuNPs z- ). The M z + complexes were chosen from previous works described in the literature and were synthesized as defined. For example, they are as follows: 1 = [RuCl(dppb)(bipy)(py)](PF6); 2 = [RuCl(dppb)(bipy)(vpy)](PF6); 3 = [RuCl(dppb)(bipy)(mepy)](PF6); 4 = [RuCl(dppb)(bipy)(tbpy)](PF6); 5 = [RuCl2(dppb)(bipy)](PF6); 6 = [Fe(bipy)3]Cl2; 7 = [Ru(bipy)3](PF6)2; 8 = [TPyP{RuCl(dppb)(bipy)}4](PF6)4; and 9 = [RuCl(p-cymene)(Diipmp)](PF6). The interactions between M z + and AuNPs z- were carried out using conductometry and UV-vis spectroscopy. These experiments allowed determination of kinetic parameters, revealing three different steps in the interaction process: induction time, flocculation, and agglomeration. The self-assembly between M z + and AuNPs z- was investigated using three different models of binding site, namely, Langmuir or direct plot, Benesi-Hildebrand, and Scatchard. These models provide the fraction of total binding sites occupied (θ), the formation constant (K f), which is dependent on the temperature and geometric structure of each group of M z +, and the Gibbs free energy of reaction (ΔG r ), which was negative for each pair of M z + and AuNPs z- , revealing a spontaneous agglomeration process. The Hill coefficient (n) was 1 for almost all complexes, indicating that agglomeration is an independent process, except for 5, where n = 2, suggesting a positive propensity to bind onto the AuNPs z- surface. The models have confirmed a noncovalent interaction between these species. The relative error in site binding does not show any variation with changes in the temperature, but a fine-tuning of the n value to 1.00 was observed with the increase of the temperature. Finally, the reduction reaction of the 4-nitrophenolate anion (4-NP-) by NaBH4 catalyzed by AuNPs z- was used in the presence of M z + as an evaluation test to show how the M z + species will disturb the 4-NP- binding site on the surface of gold nanoparticles.

Measurement of total amine site concentrations on bacterial cell surfaces using selective site-blocking and potentiometric titrations

Bacterial cell surface amine binding sites can form strong complexes with both toxic metal cations and anionic contaminants, thereby affecting the behavior of these contaminants in both natural and engineered systems. In this study, a novel approach was developed to measure the concentration of total amine sites on bacterial cell surfaces by combining selective site-blocking, potentiometric titrations and surface complexation modeling. Our controls show that the amine blocker sulfo-N-hydroxysulfosuccinimide acetate (SNHS) selectively reacts with primary and secondary amine sites but does not react with other binding sites on the bacterial cell surface. Amine site concentrations on bacterial cell surfaces, therefore, can be measured as the difference between the total site concentrations of un-blocked and SNHS-blocked bacterial samples. We measured amine site concentrations on Bacillus subtilis and Pseudomonas putida of 52 ± 5 and 78 ± 6 μmol/g, respectively, which account for 24% and 32% of the total binding sites on each bacterial surface. Our results suggest that amine sites can be present in relatively high concentrations on bacterial surfaces, and further studies that are focused on the role of cell surface amine sites on the transport and bioavailability of toxic metal cations and anionic contaminants are crucial.

Synergy of Post-Crosslinked imprinting strategy and silanol chemistry for the anisotropic construction of molecularly imprinted polymers onto Halloysite nanotubes

Searching for alternative porous substrates which can realize anisotropic preparation of “tailor-made” complementary binding sites is an important way to improve the identification efficiency and interfacial stability of the surface molecularly imprinted polymers (SMIPs). In this work, a functional polymer (FP) with multi-functional properties was firstly synthesized using 3-(isobutenyloxy) propyltrimethoxysilane, 1-(vinylbenzyl) thymidine and cinnamoyloxyethyl methacrylate (CEMA) as monomers, and then FP with post-crosslinking property was covalently deposited onto theoutersurfaceof halloysite nanotubes (HNTs) using silanol chemistry to prepare 2′-deoxyadenosine (dA) imprinted sorbents (HNTs-SMIPs). More than 65 % of dA molecules was captured within the first 2.0 h. The number of high affinity site from the total binding sites of HNTs-SMIPs is estimated to be as high as 73.25 %, and the maximum adsorption amount of HNTs-SMIPs is 48.78 μmol g -1 at 25 °C. The imprinting factor (IF) of HNTs-SMIPs towards dA is 1.89, and 79.49 % the adsorption capacity is remained after five adsorption–desorption cycles. In addition, 70.2 % of the dA from spiked human urine samples can be selectively extracted by HNTs-SMIPs. HNTs-SMIPs have excellent interfacial stability and strong affinity for the template molecule dA, and this work provides a new strategy for the specific separation and enrichment of dA.

AHoJ-DB: A PDB-wide Assignment of apo & holo Relationships Based on Individual Protein–Ligand Interactions

A single protein structure is rarely sufficient to capture the conformational variability of a protein. Both bound and unbound (holo and apo) forms of a protein are essential for understanding its geometry and making meaningful comparisons. Nevertheless, docking or drug design studies often still consider only single protein structures in their holo form, which are for the most part rigid. With the recent explosion in the field of structural biology, large, curated datasets are urgently needed. Here, we use a previously developed application (AHoJ) to perform a comprehensive search for apo-holo pairs for 468,293 biologically relevant protein–ligand interactions across 27,983 proteins. In each search, the binding pocket is captured and mapped across existing structures within the same UniProt, and the mapped pockets are annotated as apo or holo, based on the presence or absence of ligands. We assemble the results into a database, AHoJ-DB (www.apoholo.cz/db), that captures the variability of proteins with identical sequences, thereby exposing the agents responsible for the observed differences in geometry. We report several metrics for each annotated pocket, and we also include binding pockets that form at the interface of multiple chains. Analysis of the database shows that about 24% of the binding sites occur at the interface of two or more chains and that less than 50% of the total binding sites processed have an apo form in the PDB. These results can be used to train and evaluate predictors, discover potentially druggable proteins, and reveal protein- and ligand-specific relationships that were previously obscured by intermittent or partial data.Availability: www.apoholo.cz/db

Analysis of total thiol concentrations in environmental samples using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)

In this study, we developed an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) approach to measure the concentration of total reactive thiol binding sites in a range of types of environmental samples by monitoring the decrease of monobromo-(trimethylammonio)-bimane (qBBr) caused by thiol-qBBr binding reactions. We first examined the stability of qBBr and potential side reactions that could reduce the accuracy of the UHPLC-MS approach. We found that significant loss of qBBr occurs in the absence of thiols in aqueous solutions after 2–4 h due to qBBr degradation and other side reactions, such as a chloride-for-bromide substitution reaction. To address these issues, in our approach we conduct the thiol-qBBr binding reaction in the presence of excessive qBBr for only 1 h, and we compare the MS signal of unbound qBBr in a thiol-free qBBr standard with that in a thiol-bearing qBBr standard in order to calculate the decrease in the concentration of free qBBr in solution that is caused by thiol-qBBr binding. Using 100–600 nM of N-Acetyl-L-cysteine (ACYS) as a model thiol in a 2 μM qBBr standard, we found that the measured decrease in the MS signal of unbound qBBr linearly correlates to the concentration of added ACYS, with a R2 of 0.988, demonstrating that monitoring the decrease in unbound qBBr can be used to calculate the concentration of total thiol sites in a sample. We demonstrate that the new UHPLC-MS approach yields accurate results when measuring a series of single and mixed thiol standards in the 200–500 nM range, with measured values ranging from 86% to 109% of the known concentration of thiols. In addition to the above control tests, we also used the UHPLC-MS approach to measure dissolved thiol concentrations in several lake water samples. These measurements revealed that each lake contained low μM levels of total thiols, not only demonstrating the ability of the analytical approach to determine thiol site concentrations in natural waters, but also suggesting the widespread importance of these sites in controlling chalcophile element behavior in these systems. The UHPLC-MS approach not only can be used to determine the concentration of dissolved thiol sites at nM levels, but can also be adapted to measure the concentration of thiol binding sites on other environmental samples such as soil, sediment, and solid-phase organic matter surfaces.

Interfacial enzyme kinetics reveals degradation mechanisms behind resistant starch

The digestive resistance of so-called resistant starch (RS) provides an important nutritional principle. However, as yet the mechanisms underlying digestive resistance of the granular starches are not well described. We address this problem by employing a novel enzyme kinetics approach, which in contrast to conventional enzyme kinetics takes the interfacial nature of the enzyme reaction into account. Structurally different starch granules with different amylose contents were modified by the hydrolytic enzyme glucoamylase (GA). Kinetic-, adsorbtion-, imaging-, particle size-, and spectroscopic data combinedly revealed important origins of hydrolytic resistance of high-amylose starch. Our data demonstrate that amylose restricted the enzymatic catalysis efficiency on starch granules chiefly by providing less hydrolytic effective attack sites for the enzyme at granular surface. Specifically, the total binding sites (adsΓmax) (20.9 nmol/g) on starch with 0% amylose content (AC) were almost all effective attack sites (kinΓmax) (20.2 nmol/g). At 26% AC, the binding sites on the granules decreased to 13.9 nmol/g, and attack sites decreased to 7.4%. At 72% AC, the binding sites were only slightly reduced to 11.2 nmol/g, however, the attack sites were remarkably decreased to 2.6 nmol/g. Beyond the initial catalytic events, i.e., for further degraded granules, the binding and catalysis efficiency differed notably for the three starch types. At 0% AC, both binding and attack increased demonstrating increased hydrolytic susceptibility of the granules. At 26% AC only binding increased, while attack was unchanged. Interestingly, at 72% AC, binding increased, while attack deceased notably with hydrolysis time, demonstrating decreased efficiency of interfacial catalysis during the hydrolysis process.

Activating Molybdenum Carbide Nanoparticle Catalysts under Mild Conditions Using Thermally Labile Ligands.

Transition-metal carbides are promising low-cost materials for various catalytic transformations due to their multifunctionality and noble-metal-like behavior. Nanostructuring transition-metal carbides offers advantages resulting from the large surface-area-to-volume ratios inherent in colloidal nanoparticle catalysts; however, a barrier for their utilization is removal of the long-chain aliphatic ligands on their surface to access active sites. Annealing procedures to remove these ligands require temperatures greater than the catalyst synthesis and catalytic reaction temperatures and may further result in coking or particle sintering that can reduce catalytic performance. One way to circumvent this problem is by replacing the long-chain aliphatic ligands with smaller ligands that can be easily removed through low-temperature thermolytic decomposition. Here, we present the exchange of native oleylamine ligands on colloidal α-MoC1–x nanoparticles for thermally labile tert-butylamine ligands. Analyses of the ligand exchange reaction by solution 1H NMR spectroscopy, FT-IR spectroscopy, and thermogravimetric analysis–mass spectrometry (TGA-MS) confirm the displacement of 60% of the native oleylamine ligands for the thermally labile tert-butylamine, which can be removed with a mild activation step at 250 °C. Catalytic site densities were determined by carbon monoxide (CO) chemisorption, demonstrating that the mild thermal treatment at 250 °C activates ca. 25% of the total binding sites, while the native oleylamine-terminated MoC1–x nanoparticles showed no available surface binding sites after this low-temperature treatment. The mild pretreatment at 250 °C also shows distinctly different initial activities and postinduction period selectivities in the CO2 hydrogenation reaction for the ligand exchanged MoC1–x nanoparticle catalysts and the as-prepared material.

Insights into the role of extracellular DNA in heavy metal adsorption

Extracellular polymeric substances (EPS) participate in heavy metal adsorption in the aquatic environments. Extracellular DNA (eDNA) is an essential component of EPS, but its involvement in metal binding remains ambiguous. Herein, the role of eDNA in Cd(II) and Ni(II) adsorption was described using a combination of semi-quantitative and qualitative approaches. EPS were extracted from Burkholderia sp. MBR-1 and eDNA accounted for 6.9% of the total mass of EPS. The eDNA in the extracted EPS was digested using the DNase II to prepare an eDNA-free EPS sample. Potentiometric titration unveiled that the number of total binding sites of the eDNA-free EPS was 19% lower than the untreated EPS. The Cd(II) and Ni(II) adsorption capacity of the eDNA-free EPS was lower than the untreated EPS at the pH range of 4–7. At pH 7, the results of batch adsorption experiments showed that removing eDNA from EPS resulted in declines of 12.6% and 15.7% in the adsorption capacities for Cd(II) and Ni(II), respectively. Furthermore, Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy unraveled that the phosphoryl groups and purines of eDNA are responsible for Cd(II) and Ni(II) complexation. The results demonstrated that eDNA plays an essential role in heavy metal adsorption.

Sorption and transformation of arsenic by extracellular polymeric substances extracted from Synechocystis sp. PCC6803

Cyanobacteria widely distribute in the aqueous ecosystem and produce abundant extracellular polymeric substances (EPS), yet little is known about how the quantity and composition of cyanobacterial EPS change upon As exposure, and what are functions of these complex biopolymers in the As sorption and transformation processes. Here we extracted the EPS from Synechocystis sp. PCC6803, characterized their properties, quantified their components upon exposure to arsenite (As(III))/arsenate (As(V)) treatments, and investigated As binding and speciation as affected by the levels of EPS and solution pH. The total binding sites, zeta potential and reducing power of EPS were 17.47 mmol g−1, -19.72 mV and 1.71. The amounts of EPS increased by 22–65.3% and 13.8–39% when the cells were treated with 10–500 μM As(III) and As(V) respectively. The As removal was influenced by the EPS doses and solution pH, with 52.8% at pH 8.5 for As(III) and 49.5% at pH 4.5 for As(V) at 300 mg L−1 EPS. In addition, As speciation was transformed with the addition of EPS. As(V) and As(III) respectively accounted for 4.9–20.3% and 6.5–26.7% of the total dissolved As after the EPS were added (100–300 mg L−1) to the As(III) and As(V) solutions. Fourier transform infrared spectroscopy (FTIR) and three-dimensional excitation-emission fluorescence spectra (3D-EEM) revealed that As was bound to functional groups such as C═O, ─NH, and ─OH in the EPS via surface complexation/hydrophobic interactions. Taken together, this study demonstrated that the EPS extracted from Synechocystis were capable to bind and transform As and could be potentially applied to remove or detoxify As in solutions.

Influence of ultrasound on the properties of dissolved organic matter with regards to proton and metal ion binding moieties

Ultrasound is widely used in water and wastewater treatment. However, the influence of ultrasound on the composition and structure of dissolved organic matter (DOM) and consequently its behaviors is still unclear. In this study, the changes in proton and metal binding moieties in DOM caused by ultrasound at an environmentally relevant concentration (about 5 mg·L−1) were investigated using spectrophotometric titrations. The amounts of carboxylic and phenolic-like groups in DOM before and after ultrasound were quantified by the spectral parameter, Dslope350-400 (the differential slope of the Log-transformed DOM absorbance in the wavelength region of 350–400 nm) in combination with the non-ideal competitive adsorption (NICA) model. The results demonstrate that the concentration of carboxylic-like sites decreased with an increase in the ultrasound intensity at the same reaction time. However, the concentration of phenolic-like sites increased with an increase in the ultrasound intensity in most cases. The amounts of bound metal Ca(II) and Al(III) ions decreased with an increase of the ultrasound intensity due to the reduction of available total binding sites (carboxylic-like and phenolic-like groups) in the DOM after ultrasound. This study provides detailed in-situ information concerning the changes in the properties and behaviors of DOM in ultrasound, which would be useful to understand in more detail and to optimize the utility of ultrasound in water and wastewater treatment.

Optical property of dissolved organic matters (DOMs) and its link to the presence of metal ions in surface freshwaters in China

It is believed that dissolved organic matter (DOM) plays an important role in carrying metal ions through global cycle and distribution, while this point has not yet been elucidated clearly as DOM is extremely heterogeneous and site-specific. In this study, optical properties of DOM in 53 surface freshwater sites all over China were recorded by UV–Vis absorbance spectroscopy in pH range 3–11. The subtle processes of protonation-deprotonation of DOM were quantified by tracking the changes of DOM spectra. The binding capacities of DOMs-binding sites and affinity constants-were interpreted by introducing a spectral parameter, differential log-transformed differential absorbance at 400 nm (DlnA400) in combination with Non-Ideal Competitive Adsorption (NICA) Model. It is found that the presence of dissolved Zn, Hg and Ni in the examined waters show strong correlation with total binding sites in DOMs. However, the presence of some other metals with high affinities to DOM, e.g. Cu, Fe and so on, have not demonstrated strong correlation with the total binding sites in DOMs. This indicates the presence of these metals may be seriously influenced by other factors besides DOM. This study demonstrates that the spectroscopic titration approach could potentially provide more structure-specific in situ information about DOM and help to understand the role of DOM in the speciation and bioavailability of toxic metal ions.

Turmeric Powder as a Natural Heavy Metal Chelating Agent: Surface Characterisation

The present study was conducted to investigate surface properties of turmeric in order to evaluateits detoxification potential and ability to sequester toxic metals ions. Scanning Electron Microscopy (SEM),Energy Dispersion Spectroscopy (EDS), Infra-Red (IR) spectroscopy and potentiometric titrations wereemployed for characterisation of the surface of turmeric powder. Spectroscopic studies revealed that thesurface of turmeric powder was porous mainly composed of polymeric -OH , -NH , -CH2 , -COO and-OH groups of polysaccharides. From potentiometric titrations and modelling of batch titration data, it wasfound that surface of the turmeric contains at least four binding sites with pKa values 3.56 (pK1), 4.83(pK2), 7.68 (pK3) and 10.4 (pK4). Turmeric powder contains highest concentration of amino and hydroxylgroups for the pK4 values i.e., 0.55 mmol/ g. The total binding sites concentration for turmeric powderwas 1.2 mmol/ g.

Influence of sulfhydryl sites on metal binding by bacteria

The role of sulfhydryl sites within bacterial cell envelopes is still unknown, but the sites may control the fate and bioavailability of metals. Organic sulfhydryl compounds are important complexing ligands in aqueous systems and they can influence metal speciation in natural waters. Though representing only approximately 5–10% of the total available binding sites on bacterial surfaces, sulfhydryl sites exhibit high binding affinities for some metals. Due to the potential importance of bacterial sulfhydryl sites in natural systems, metal-bacterial sulfhydryl site binding constants must be determined in order to construct accurate models of the fate and distribution of metals in these systems. To date, only Cd-sulfhydryl binding has been quantified.In this study, the thermodynamic stabilities of Mn-, Co-, Ni-, Zn-, Sr- and Pb-sulfhydryl bacterial cell envelope complexes were determined for the bacterial species Shewanella oneidensis MR-1. Metal adsorption experiments were conducted as a function of both pH, ranging from 5.0 to 7.0, and metal loading, from 0.5 to 40.0μmol/g (wet weight) bacteria, in batch experiments in order to determine if metal-sulfhydryl binding occurs. Initially, the data were used to calculate the value of the stability constants for the important metal-sulfhydryl bacterial complexes for each metal-loading condition studied, assuming a single binding reaction for the dominant metal-binding site type under the pH conditions of the experiments. For most of the metals that we studied, these calculated stability constant values increased significantly with decreasing metal loading, strongly suggesting that our initial assumption was not valid and that more than one type of binding occurs at the assumed binding site. We then modeled each dataset with two distinct site types with identical acidity constants: one site with a high metal-site stability constant value, which we take to represent metal-sulfhydryl binding and which dominates under low metal loading conditions, and another more abundant site that we term non-sulfhydryl sites that becomes important at high metal loadings. The resulting calculated stability constants do not vary significantly as a function of metal loading and yield reasonable fits to the observed adsorption behaviors as a function of both pH and metal loading. We use the results to calculate the speciation of metals bound by the bacterial envelope in realistic bacteria-bearing, heavy metal contaminated systems in order to demonstrate the potential importance of metal-sulfhydryl binding in the budget of bacterially-adsorbed metals under low metal-loading conditions.

Half Metallic Transition In Silver-adsorbed Zigzag Graphene Nanoribbons

We investigate Ag atom bonding to zigzag graphene ribbons and properties of these systems to increase the understanding of spin transport. Results concerning the total energies preferred binding sites, equilibrium distances, and electronic character for Ag adatoms on ZGNR are predicted in this study. The study also provides insight into the diffusion kinetics of adsorbed Ag atoms on zigzag edged graphene nanoribbons. The ribbons considered in the present work have an antiferromagnetic ground state and undergoes transition from semiconducting to half-metallic on spin polarization. DOS profiles of Ag-adsorbed ZGNR at the bridge site reveals low DOS across the Fermi level ensuring its semiconducting character, validated from the unpolarized spin calculations. Though nonlinear at biases less than 1V in the FM and AFM calculations, the spin current of atop adsorbed ribbon increases rapidly with the increase of the bias voltage beyond 1V. Copyright © 2016 VBRI Press.

Bacterial superglue enables easy development of efficient virus-like particle based vaccines.

BackgroundVirus-like particles (VLPs) represent a significant advance in the development of subunit vaccines, combining high safety and efficacy. Their particulate nature and dense repetitive subunit organization makes them ideal scaffolds for display of vaccine antigens. Traditional approaches for VLP-based antigen display require labor-intensive trial-and-error optimization, and often fail to generate dense antigen display. Here we utilize the split-intein (SpyTag/SpyCatcher) conjugation system to generate stable isopeptide bound antigen-VLP complexes by simply mixing of the antigen and VLP components.ResultsGenetic fusion of SpyTag or SpyCatcher to the N-terminus and/or C-terminus of the Acinetobacter phage AP205 capsid protein resulted in formation of stable, nonaggregated VLPs expressing one SpyCatcher, one SpyTag or two SpyTags per capsid protein. Mixing of spy-VLPs with eleven different vaccine antigens fused to SpyCatcher or SpyTag resulted in formation of antigen-VLP complexes with coupling efficiencies (% occupancy of total VLP binding sites) ranging from 22–88 %. In mice, spy-VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly increased antibody titer, affinity, longevity and functional efficacy compared to corresponding vaccines employing monomeric proteins. The spy-VLP vaccines also effectively broke B cell self-tolerance and induced potent and durable antibody responses upon vaccination with cancer or allergy-associated self-antigens (PD-L1, CTLA-4 and IL-5).ConclusionsThe spy-VLP system constitutes a versatile and rapid method to develop highly immunogenic VLP-based vaccines. Our data provide proof-of-concept for the technology’s ability to present complex vaccine antigens to the immune system and elicit robust functional antibody responses as well as to efficiently break B cell self-tolerance. The spy-VLP-system may serve as a generic tool for the cost-effective development of effective VLP-vaccines against both infectious- and non-communicable diseases and could facilitate rapid and unbiased screening of vaccine candidate antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0181-1) contains supplementary material, which is available to authorized users.

Chemical and structural characterization of copper adsorbed on mosses (Bryophyta)

The adsorption of copper on passive biomonitors (devitalized mosses Hypnum sp., Sphagnum denticulatum, Pseudoscleropodium purum and Brachythecium rutabulum) was studied under different experimental conditions such as a function of pH and Cu concentration in solution. Cu assimilation by living Physcomitrella patents was also investigated. Molecular structure of surface adsorbed and incorporated Cu was studied by X-ray Absorption Spectroscopy (XAS). Devitalized mosses exhibited the universal adsorption pattern of Cu as a function of pH, with a total binding sites number 0.05–0.06mmolgdry−1 and a maximal adsorption capacity of 0.93–1.25mmolgdry−1 for these devitalized species. The Extended X-ray Absorption Fine Structure (EXAFS) fit of the first neighbor demonstrated that for all studied mosses there are ∼4.5 O/N atoms around Cu at ∼1.95Å likely in a pseudo-square geometry. The X-ray Absorption Near Edge Structure (XANES) analysis demonstrated that Cu(II)-cellulose (representing carboxylate groups) and Cu(II)-phosphate are the main moss surface binding moieties, and the percentage of these sites varies as a function of solution pH. P. patens exposed during one month to Cu2+ yielded ∼20% of Cu(I) in the form of Cu–S(CN) complexes, suggesting metabolically-controlled reduction of adsorbed and assimilated Cu2+.

Albumin Binding Function: The Potential Earliest Indicator for Liver Function Damage.

Background. Currently there is no indicator that can evaluate actual liver lesion for early stages of viral hepatitis, nonalcoholic fatty liver disease (NAFLD), and cirrhosis. Aim of this study was to investigate if albumin binding function could better reflect liver function in these liver diseases. Methods. An observational study was performed on 193 patients with early NAFLD, viral hepatitis, and cirrhosis. Cirrhosis patients were separated according to Child-Pugh score into A, B, and C subgroup. Albumin metal ion binding capacity (Ischemia-modified albumin transformed, IMAT) and fatty acid binding capacity (total binding sites, TBS) were detected. Results. Both IMAT and TBS were significantly decreased in patients with NAFLD and early hepatitis. In hepatitis group, they declined prior to changes of liver enzymes. IMAT was significantly higher in cirrhosis Child-Pugh class A group than hepatitis patients and decreased in Child-Pugh class B and class C patients. Both IMAT/albumin and TBS/albumin decreased significantly in hepatitis and NAFLD group patients. Conclusions. This is the first study to discover changes of albumin metal ion and fatty acid binding capacities prior to conventional biomarkers for liver damage in early stage of liver diseases. They may become potential earliest sensitive indicators for liver function evaluation.

The Effects of Bacterial Surface Adsorption and Exudates on HgO Precipitation

abstractThe effects of nonmetabolic bacterial cell wall adsorption and the presence of bacterial exudates on the precipitation of mineral phases from solution is not well constrained experimentally. In this study, we measured the extent of Hg(II) removal from solution, in the presence and absence of nonmetabolizing cells of Bacillus subtilis in both Cl-free and Cl-bearing systems with Hg concentrations ranging from undersaturation to supersaturation with respect to montroydite [HgO(s)]. Total Hg molalities ranged from 10−5.00 to 10−2.00 M at pH 4.50 and 7.00; the ionic strength of the experiments was kept constant using 0.01 M NaClO4, and the wet mass of bacteria was held constant at 5 g/L for each biotic experiment.The biotic systems exhibited enhanced Hg(II) removal from solution relative to the abiotic controls in undersaturated conditions. However, thermodynamic modeling of the experimental systems strongly suggests that all of this Hg removal can be ascribed to Hg adsorption onto cell envelope functional groups. There was no evidence for enhanced Hg removal due to precipitation in bulk solutions that were undersaturated with respect to the solid phase. Under the highest total Hg concentrations studied in both the Cl-free and Cl-bearing systems, bacteria inhibit precipitation, maintaining high concentrations of Hg in solution. Cell-free, exudate-bearing control experiments suggest that aqueous complexation between Hg and the bacterially-produced exudates accounts for at least some of the precipitation inhibition. However, a comparison of total available binding sites on the exudates with the concentration of Hg in solution suggests that aqueous complexation alone can not account for the observed elevated final aqueous Hg concentrations in solution, and that the exudates likely exert a kinetic inhibition on the precipitation reaction as well.

Spectrophotometric Determination of Affinities of α‐Factors for Their G Protein‐Coupled Receptors in Saccharomyces cerevisiae

A new and relatively simple spectrophotometric technique has been developed for the accurate determination of α‐factor pheromone affinities for Ste2p whole cell receptor in yeast a‐cells. We designed and tested nine detector peptides containing mono‐ (ε412 = 14 500) or tri‐cysteine residues (ε412 = 43 660). The free unbound detector was detected using Ellman's reagent at 412 nm. Saturation binding studies using Saccharomyces cerevisiae Y 7925 ( MATa ) at a concentration of 2.5 × 1011 cells/mL with the highest affinity detector 1, [Orn6]α‐factor‐[Cys]3, resulted in a dissociation constant ( K D ) of 1.67 × 10−7 and total binding sites per cell (B cell = 29 500 sites/cell) comparable with those obtained using radiolabeled binding assays. Competitive binding assay using five nonchromogenic α‐factor analogs allowed for the determination of each K D value. [Orn6,d‐Ala9]α‐factor showed the highest receptor affinity ( K D = 1.03 × 10−7 M), which was threefold higher than that of native α‐factor. This assay provides rapid and convenient results for determining the relative affinities of nonchromogenic α‐factors and eliminates the need for radioactive waste disposal.

Confirmation of the validity of the current characterization of immunochemical reactions by kinetic exclusion assay

Prior observations that questioned the validity of kinetic exclusion assays were based on the mistaken assumption that the assays quantified the fraction of those antibody molecules that had unoccupied binding sites. Instead, the standard KinExA assay quantifies the fraction of total antibody binding sites that are unoccupied, regardless of the number of unoccupied sites on each antibody molecule. Although the standard KinExA analysis assumes that there is only a small probability of antibody-site capture by the affinity matrix, the results of numerical simulations demonstrate the reliability of dissociation constants obtained by the standard KinExA analysis for capture probabilities as high as 30%. This finding further strengthens the potential of kinetic exclusion assays as the procedure of choice for the rapid and accurate characterization of immunochemical reactions that forms part of screening processes in the search for therapeutic antibodies.