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1
E., Subramanian, Murugesan R., and Anitha G. "Molecular discriminative binding behaviour of crosslinked polyvinylpyrrolidone with analogous dyes orange G and orange H." Journal of Indian Chemical Society Vol. 80, Oct 2003 (2003): 894–98. https://doi.org/10.5281/zenodo.5839300.
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Department of Chemistry, Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli-627 012, India <em>E-mail : </em>smanian2002@yahoo.com <em>Fax : </em>91-462-2334363/2322973 <em>Manuscript received 15 March 2002, revised 1 October 2002, accepted 23 May 2003</em> Two structurally analogous dyes Orange G (OG) and Orange H (OH) with slight difference in their molecular features (in the number and position of sulfonato groups) were studied for their binding to crosslinked polyvinylpyrrolidone (CPVP) in double-distilled water (pH =5.5) and phosphate buffer (pH = 7.2) at different temperatures by batch adsorption technique. The binding data were analysed by Scatchard method and Giles adsorption isotherm. CPVP clearly exposed the differing molecular features of associating dyes through remarkable difference in binding plots. OG showed a lesser degree of binding than did OH in both the solvent media but the difference became small in buffer. Further, OG displayed a binding mode of mixed cooperativity with the likely formation of two succeeding complexes while OH sorbed with a simple positive cooperativity forming only one type of complex.
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Yi, Li Na, Xiao Ying Yin, Yi Fan Jiang, and Qing Shan Liu. "Preparation and Properties of Ginsenoside Rg1 Molecularly Imprinted Polymers." Advanced Materials Research 550-553 (July 2012): 1715–18. http://dx.doi.org/10.4028/www.scientific.net/amr.550-553.1715.
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Molecularly imprinted polymers (MIPs) were prepared by precipitation polymerization with ginsenoside Rg1 as the template molecule. The morphology of MIPs was characterized by scanning electronmicroscope (SEM) and its static adsorption capacity was measured by the Scatchard equation. Scatchard analysis revealed that the homogeneous binding sites were formed in the polymers. The application of MIPs with high affinity toward the template molecule might offer a novel method for the enrichment and determination of active compounds in the traditional herbal medicine.
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Armatas, G. S., D. E. Petrakis, and P. J. Pomonis. "A method of distinction between microporosity and mesoporosity using BET–Scatchard plots." Microporous and Mesoporous Materials 83, no. 1-3 (2005): 251–61. http://dx.doi.org/10.1016/j.micromeso.2005.05.005.
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Delforge, Jacques, André Syrota, Michel Bottlaender, et al. "Modeling Analysis of [11C]Flumazenil Kinetics Studied by PET: Application to a Critical Study of the Equilibrium Approaches." Journal of Cerebral Blood Flow & Metabolism 13, no. 3 (1993): 454–68. http://dx.doi.org/10.1038/jcbfm.1993.60.
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The multi-injection modeling approach was used for the in vivo quantitation of benzodiazepine receptors in baboon brain using positron emission tomography (PET) and [11C]flumazenil (RO 15-1788) as a specific ligand. The model included three compartments (plasma, free, and bound ligand) and five parameters (including the benzodiazepine receptor concentration). The plasma concentration after correction for the metabolites was used as the input function. The experimental protocol consisted of four injections of labeled and/or unlabeled ligand. This protocol allows the evaluation, from a single experiment, of the five model parameters in various regions of interest. For example, in the temporal cortex, the concentration of receptor sites available for binding ( B′max) and the equilibrium dissociation constant ( Kd) were estimated to be 70 ± 15 pmol/ml and 15.8 ± 2.2 n M, respectively. The validity of the equilibrium approach, which is the most often used quantitation method, has been studied from simulated data calculated using these model parameters. The equilibrium approaches consist of reproducing in PET studies the experimental conditions that permit the use of the usual in vitro methods such as Scatchard analysis. These approaches are often open to criticism because of the difficulty of defining the notion of equilibrium in in vivo studies. However, it appears that the basic relation of Scatchard analysis is valid over a broader range of conditions than those normally used, such as the requirement of a constant bound/free ratio. Simulations showed that the values of the receptor concentration ( B′max) and the equilibrium dissociation constant ( Kd) found using Scatchard analysis are always underestimated. These simulations also suggest an explanation concerning the dependency of B′max and Kd on the time point employed for the Scatchard analysis, a phenomenon found by several authors. To conclude, we propose new protocols that allow the estimation of the B′max and Kd parameters using a Scatchard analysis but based on a protocol including only one or two injections. These protocols being entirely noninvasive, it thus becomes possible to investigate possible changes in receptor density and/or affinity in patients.
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Liu, Qing Shan, Ke Qin Li, Jun Li, Xiao Ying Yin, and Tian Hua Yan. "A Novel Method for Prepairing Higher Performance Picroside I Surface Molecularly Imprinted Polymers for TCM Research." Advanced Materials Research 785-786 (September 2013): 642–45. http://dx.doi.org/10.4028/www.scientific.net/amr.785-786.642.
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To establish a novel method for preparing molecularly imprinted polymers for Picroside I with better performance on TCM research contrast to previous studies, we have prepared novel surface molecular imprinted polymers (S-MIPs) using Picroside I as the template molecule, Acrylamide (AM) as the functional monomer, and silica gel as the carrier. The morphology of S-MIPs was characterized by scanning electron microscope (SEM) and its static adsorption capacity was measured by the Scatchard equation.
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Marczyński, Zbigniew, Marian Mikołaj Zgoda, Andrzej Stańczak, Sławomira Nowak, Jerzy Jambor та Beata Skibska. "Predicted real solubility (– log x2) and the level of hydrophilic-lipophilic balance (HLBRequ.) of phytochemicals contained in extracts isolated from linden inflorescence (Tiliae flos) with solvents of diversified polarity (εM)". Herba Polonica 65, № 3 (2019): 39–50. http://dx.doi.org/10.2478/hepo-2019-0017.
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Summary Introduction: The broad spectrum of pharmacological properties of linden inflorescence extracts results from polarity and the level of hydrophilic-lipophilic balance of solvents (medium) used to separate compatible phytochemical structures with the expected pharmacotherapeutic profile. Objective: The use of the general Hildebrand-Scatchard-Fedors theory of solubility to calculate the predicted solubility of classes of phytochemicals contained in linden inflorescences (Tiliae flos) and the indication of those structures which, due to their high solubility in the medium, are responsible for the profile of pharmacological activity. Material and methods: The Hildebrand, Scatchard equation, supported with computational technique proposed by Fedors, allows calculation of the solubility parameters of the extraction medium. Despite application reservations, it is a fundamental tool for estimating the predictable solubility of phytochemicals in real solution. Results: The structure of phytochemicals isolated from linden inflorescences (Tiliae flos) owing to the use of solvents of significantly diversified polarity (–dielectric constant – εM) was the basis for calculating the molar evaporation energy – ΣΔEi (cal/mol) and molar volume – ΣΔVi (cm3/mol) by Fedors method, which are fundamental quantities necessary to estimate the solubility parameter – δ1/2 and required solubility level of hydrophilic-lipophilic balance – HLBRequ. Conclusions: Results of the presented research indicate that basing on the parameters characterizing the structure of phytochemicals and the calculated ideal (–logxi2) and predicted real (– log x2) solubility, it is possible – using the general Hildebrand-Scatchard-Fedors theory of solubility – to choose selectively the cascade of extraction media in order to distinguish in the plant material chemical and structural individuals of different polarity.
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Liu, Qing Shan, Li Na Yi, Qiu Juan Wang, Qing Long Guo, Yi Fan Jiang, and Xiao Ying Yin. "A Novel Method for Preparing the Surface Molecularly Imprinted Polymers to Target Isolate Ginsenoside Rg1 and its Analogues." Advanced Materials Research 535-537 (June 2012): 2400–2403. http://dx.doi.org/10.4028/www.scientific.net/amr.535-537.2400.
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To establish a novel method for preparing molecularly imprinted polymers for ginsenoside Rg1 with better character contrast to previous studies, we have prepared novel surface molecular imprinted polymers (S-MIPs) using ginsenoside Rg1 as the template molecule, Acrylamide (AM) as the functional monomer, and silica gel as the carrier. The morphology of S-MIPs was characterized by scanning electron microscope (SEM) and its static adsorption capacity was measured by the Scatchard equation.
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Zhou, Xing Nong, Yao Yu, Song Liu, Shou Lei Yan, Qing Zhang Wang, and Jie Li. "Preparation and Recognized Characteristic of Histamine Molecularly Imprinted Polymers." Advanced Materials Research 550-553 (July 2012): 780–86. http://dx.doi.org/10.4028/www.scientific.net/amr.550-553.780.
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HA MIP was prepared in acetonitrile-ethylene glycol mixed solvent ( 20:1，v/v), HA was used as the template, methacrylic acid (MAA) as the functional monomer, azobisisobutyronitrile (AIBN) as the initiator and ethylene glycol dimethaerylate (EGDMA) as the cross-linker. The UV spectrophotometry was used to demonstrate the interaction between HA and MAA. The adsorption characteristics of MIP to HA have been studied by equilibrium binding experiment and Scatchard analysis. The data obtained show that MIP reached equilibrium within 6 h. It is found that within the studied concentration range one HA molecule is entrapped by two MAA molecules The Scatchard chart shows the apparent maximum binding capacity (Bmax) and the dissociation contents (KD) of MIP are 170.5 μmol/g and 0.18 mmol/L, respctively. The MIP synthesized by this method have better binding ability to histamine and can be applied on the separation and detection of histamine.
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Frąckowiak, Teresa, Tomasz Bączek, Roman Kaliszan та ін. "Binding of an Oxindole Alkaloid from Uncaria tomentosa to Amyloid Protein (Aβ1-40)". Zeitschrift für Naturforschung C 61, № 11-12 (2006): 821–26. http://dx.doi.org/10.1515/znc-2006-11-1209.
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Abstract The primary aim of this work was to determine the interactions of an oxindole alkaloid (mitraphylline) isolated from Uncaria tomentosa with β-amyloid 1-40 (Aβ1-40 protein) applying the capillary electrophoresis (CE) method. Specifically the Hummel-Dreyer method and Scatchard analysis were performed to study the binding of oxindole alkaloids with Aβ1-40 protein. Prior to these studies extraction of the alkaloid of interest was carried out. Identification of the isolated alkaloid was performed by the use of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) combined with electrospray ionization mass spectrometry (ESI-MS). The proposed approach was proved to be an efficient and accurate method for specific compound isolation and identification purposes. Moreover, analytical information from the CE approach can be considered as the valuable tool for binding constant determination. The binding constant of mitraphylline with Aβ1-40 protein determined by the Hummel-Dreyer method and Scatchard analysis equals K = 9.95 x 105 m-1. The results obtained showed the significant binding of the tested compound with Aβ1-40 protein. These results are discussed and interpreted in the view of developing a strategy for identification of novel compounds of great importance in Alzheimer disease therapy.
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Huang, Ying, Yan Xiong, Zhong Bin Ye, and Zhu Jun Zhang. "Flow Injection on Line Oxidizing Fluorometry Coupled to Microdialysis Sampling for Studying Phentolamine-Bovine Serum Albumin Interaction." Advanced Materials Research 554-556 (July 2012): 2093–97. http://dx.doi.org/10.4028/www.scientific.net/amr.554-556.2093.
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Determination of phentolamine-bovine serum albumin (BSA) interaction based on a flow-injection microdialysis sampling fluorescence system (FI-MD-FL) was proposed. The analytical procedure was based on the oxidation of phentolamine by acidic Ce(IV) and monitoring of the fluorescence intensity of the formed Ce(III). The drug of phentolamine and BSA were mixed in different molar ratios in Ringer’s solution and incubated in a water bath. The microdialysate samples were on-line merged with acidic Ce(IV) solution by putting a microdialysis probe into the mixed solution and perfused with Ringer’s solution at 10 μL min-1. Then the sample was reached the flow cell, excited and monitored at 256/355nm (λex/λem). The dialytic efficiency under the experimental conditions was 38.8±2.2% (n=3). The data obtained was analyzed with Scatchard analysis and Klotz plot. The estimated association constant (K) and the number of the binding site (n) on one molecule of BSA by Scatchard analysis were 1.78×105 L mol-1 and 0.69, respectively. The proposed method has been applied to the study of the binding of phentolamine to bovine serum albumin (BSA) in vitro. The method provided a fast and simple technique for the study of drug-protein interactions.
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Liu, Qing Shan, Li Na Yi, Ke Qin Li, and Xiao Ying Yin. "A Novel Extraction Method for Active Compounds from Traditional Chinese Medicinal Materials Environment Friendly." Advanced Materials Research 726-731 (August 2013): 618–21. http://dx.doi.org/10.4028/www.scientific.net/amr.726-731.618.
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A novel extraction method based on environmental protection was founded through molecularly imprinted polymers (MIPs), overcoming the defects of traditional extraction methods, such as reagents consuming, complex operation and environmental pollution. MIPs were prepared by precipitation polymerization with picroside II or ginsenoside rg1 as the template molecule. The morphology of MIPs was characterized by scanning electron microscope and its static adsorption capacity was measured by the Scatchard equation. In addition, MIPs were filled in the solid phase extraction (SPE) column to separate and enrich the template molecule and its analogues, compared with C18-SPE column. The results indicated that MIPs have high affinity toward the template molecules, which might offer a novel method for the extraction of active compounds in the traditional herbal medicine.
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Kienhuis, C. B., J. J. Heuvel, H. A. Ross, L. M. Swinkels, J. A. Foekens, and T. J. Benraad. "Six methods for direct radioiodination of mouse epidermal growth factor compared: effect of nonequivalence in binding behavior between labeled and unlabeled ligand." Clinical Chemistry 37, no. 10 (1991): 1749–55. http://dx.doi.org/10.1093/clinchem/37.10.1749.
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Abstract Mouse epidermal growth factor (EGF) was radioiodinated by six different direct iodination methods. The 125I-labeled EGF preparations were distinguished by analyzing the binding of the radioligand to the EGF receptor (EGFR)-containing human placental membranes. The receptor-binding affinity of EGF labeled with Chloramine T was less than the affinity of unlabeled EGF, which precluded an accurate determination of the specific radioactivity of the 125I-labeled EGF preparation by "self-displacement analysis." Scatchard analysis of competitive binding data (increasing concentrations of unlabeled EGF) obtained with commercially prepared 125I-labeled EGF (Chloramine T method), according to the specific radioactivity stated by the manufacturer, resulted in a substantial underestimation of the apparent number of receptors. Iodination of EGF with Iodogen or Iodo-beads, reagents claimed to be more gentle because of their solid state, also yielded 125I-labeled EGF preparations that were not equivalent to the native EGF in receptor binding. In contrast, equivalence in the ligand-receptor interaction between labeled and unlabeled EGF could be achieved by iodinating EGF with iodine monochloride (ICl), Protag-125, or lactoperoxidase-glucose oxidase-coupled beads (Enzymobeads). Scatchard plots of saturation and competitive binding data obtained with these 125I-labeled EGF preparations produced identical results for apparent receptor number and apparent dissociation constants. Such radioiodinated EGF preparations yield relevant binding data in competition studies of labeled and unlabeled EGF.
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Akpomie, Kovo G., Anthony C. Ofomatah, Helen O. Chukwuemeka-Okorie, et al. "Equilibrium isotherm investigation on the sequestration of ciprofloxacin from solution via adsorption onto yam peel powder." IOP Conference Series: Earth and Environmental Science 1178, no. 1 (2023): 012020. http://dx.doi.org/10.1088/1755-1315/1178/1/012020.
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Abstract In this work, the adsorption of ciprofloxacin onto yam peel biosorbent was studied by batch method. The equilibrium isotherm analysis of the adsorption process was evaluated to provide insight into the removal mechanism. A decrease in the percentage removal (75.0 – 60.8%) and an increase in adsorption capacity (6.0 – 24.3 mg/g) with an increase in ciprofloxacin concentration from 20 – 100 mg/L was obtained. The isotherm was analyzed by the Langmuir, Temkin, Freundlich, and Scatchard models, and the best fit was obtained for the Freundlich model with a R2 of 0.9918. The separation factor in the range of 0.238 – 0.609 and the Freundlich adsorption intensity of 1.492 indicated a favorable adsorption of ciprofloxacin on yam peel. A monolayer adsorption capacity of 42.81 mg/g was obtained for yam peel which was higher than other efficient adsorbents. The Scatchard model gave a linear fit to the uptake data with R2 of 0.9653 and sum square error of 0.008. The isotherm analysis revealed complex adsorption involving multi mechanisms in the overall process. The results of this investigation showed that yam peel could be utilized as an efficient agricultural waste for the adsorption of ciprofloxacin from wastewater.
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Good, M. J., W. J. Hage, C. L. Mummery, S. W. De Laat, and J. Boonstra. "Localization and quantification of epidermal growth factor receptors on single cells by confocal laser scanning microscopy." Journal of Histochemistry & Cytochemistry 40, no. 9 (1992): 1353–61. http://dx.doi.org/10.1177/40.9.1506672.
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We have established a method for quantifying binding of fluorescence-labeled growth factors to their receptors on single cells in situ with the confocal laser scanning microscope (CLSM). Biotinylated epidermal growth factor (EGF) coupled to phycoerythrin-labeled anti-biotin was used to compare the levels of fluorescence on three different cell types for which the number of EGF factors was known from Scatchard analysis of [125I]-EGF binding. The results showed that as few as 10,000 receptors/cell were detectable above back-ground. This method will provide a rapid and quantifiable alternative to autoradiography for ligand binding to single cells in situ.
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Prystay, Linda, Mylene Gosselin, and Peter Banks. "Determination of Equilibrium Dissociation Constants in Fluorescence Polarization." Journal of Biomolecular Screening 6, no. 3 (2001): 141–50. http://dx.doi.org/10.1177/108705710100600304.
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A simple mathematical model (the FP Kd model) is used to generate the dissociation equilibrium constant (Kd) for G protein-coupled receptor-ligand binding measured using fluorescence polarization (FP) saturation curve analysis. The model generates data that may be analyzed by the method of Scatchard. The validity of the FP Kd model is proven in six model systems in which the modeled Kd values are within a factor of 5 of inhibitory equilibrium constant values obtained from radioligand competition assays.
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Zhang, Yan, Xingming Wang, and Lisheng Ding. "Interaction between tryptophan-vanillin Schiff base and herring sperm DNA." Journal of the Serbian Chemical Society 75, no. 9 (2010): 1191–201. http://dx.doi.org/10.2298/jsc100128107z.
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The interaction of the Schiff base (K[HL]) with herring sperm DNA was studied by UV-vis absorption, fluorescence and viscosity methods in a physiological pH environment (pH 7.40), where the Schiff base was derived from vanillin and L-tryptophan. A binding ratio of nK[HL]:nDNA = 5:1 and an apparent molar absorption coefficient of ?K[HL]-DNA = 4.98 ? 105 L?mol-1?cm-1 were confirmed by the mole ratio method. The binding constants of KB ? 28?C = 1.94 ? 105 L?mol-1 and KB ? 37?C = 1.09 ? 105 L?mol-1 were obtained by the double reciprocal method. Thermodynamic parameters suggest that the interaction between K[HL] and DNA is driven mainly by enthalpy. Combined with Scatchard methods and viscosity methods, the results indicate the presence of intercalation and groove binding between K[HL] and DNA.
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Durán-Merás, Isabel, Arsenio Muñoz De La Peña, Francisco Salinas та Isabel Rodríguez Cáceres. "Spectrofluorimetric Study of the Inclusion Complex of 7-Hydroxymethylnalidixic Acid with γ-Cyclodextrin in Aqueous Solution". Applied Spectroscopy 51, № 5 (1997): 684–88. http://dx.doi.org/10.1366/0003702971940828.
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A fluorimetric study on the spectral characteristics and acid-base behavior of 7-hydroxymethylnalidixic acid has been performed. The host–guest complexation process between γ-cyclodextrin and 7-hydroxymethylnalid ixic acid has also been investigated by fluorescence spectroscopy. Scatchard and Benesi-Hildebr and methods have been applied to determine the stoichiometry of the complex, and a 1:1 stoichiometric ratio has been established. A K1 = 118 ± 25 M−1 formation constant has been calculated for the inclusion complex, by using the changes produced on the fluorescence of 7-hydroxymethylnalid ixic acid, when it is included on the hydrophobic cyclodextrin cavity. A fluorimetric method of determination of 7-hydroxymethylnalid ixic acid, in γ-cyclodextrin aqueous solution, has been proposed. The analytical parameters and detection and quantification limits of the method have been determined.
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Geng, Ping, Qing Shan Liu, Kebaituli Gulibanumu, Xu Li, Ke Qin Li, and Xiao Ying Yin. "Practice and Synthetize Molecularly Imprinted Polymers for Seperating Target Neuro-Protective Compounds from TCM with Less Pollution." Advanced Materials Research 955-959 (June 2014): 239–42. http://dx.doi.org/10.4028/www.scientific.net/amr.955-959.239.
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Molecularly imprinted polymers (MIPs) are synthetic materials that can be the environmental protection extraction method in TCM research and industry. They can overcome the defects of traditional extract methods and environmental pollution. In our research, MIPs were prepared by precipitation polymerization with neuro-protective picroside I and ginsenoside Rb1 as the template molecule. Moreover, the morphology of MIPs was characterized by electron microscope scanning and the static adsorption capacity was measured by the Scatchard equation. Finally, MIPs were made into MIP-SPE columns to enrich the template molecule and its analogues comparing with C18-SPE column and the results show that MIPs have good affinity and selectivity towards the Rb1 and Picroside I in SPE columns. This research may offer a more environmentally friendly method to extract active compounds in the traditional herbal.
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FUCHS, Hendrik, and Reinhard GESSNER. "The result of equilibrium-constant calculations strongly depends on the evaluation method used and on the type of experimental errors." Biochemical Journal 359, no. 2 (2001): 411–18. http://dx.doi.org/10.1042/bj3590411.
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The determination of equilibrium constants is a widespread tool both to understand and to characterize protein–protein interactions. A variety of different methods, among them Scatchard analysis, is used to calculate these constants. Although more than 1000 articles dealing with equilibrium constants are published every year, the effects of experimental errors on the results are often disregarded when interpreting the data. In the present study we theoretically analysed the effect of various types of experimental errors on equilibrium constants derived by three different methods. A computer simulation clearly showed that certain experimental errors, namely inaccurate background correction, inexact calibration, saturation effects, slow kinetics and simple scattering, can adversely affect the result. The analysis further revealed that, for a given type of error, the same data set can produce different results depending on the method used.
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Kohlhepp, Sue J., David N. Gilbert, and James E. Leggett. "Influence of Assay Methodology on the Measurement of Free Serum Ceftriaxone Concentrations." Antimicrobial Agents and Chemotherapy 42, no. 9 (1998): 2259–61. http://dx.doi.org/10.1128/aac.42.9.2259.
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ABSTRACT The influence of assay methodology on the measurement of the active free fraction of ceftriaxone in plasma was determined. The free fraction was measured by three methods: agar diffusion bioassay, precipitation of plasma protein with methanol followed by high-performance liquid chromatography (HPLC) of the supernatant, and ultrafiltration of plasma followed by HPLC of the filtrate. In human serum, the free ceftriaxone levels were significantly lower (P = 0.03) when measured on ultrafiltrates compared to the other two methods. This difference disappeared when dolphin serum was studied. After ultrafiltration, human serum was shown, by Scatchard plot analysis, to have two ceftriaxone binding sites. Species differences were also demonstrated. Hence, in humans, determination of free plasma ceftriaxone varies with the assay method employed.
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Balikungeri, Antoine. "Acid-Base Properties of 2-Morpholinoethanesulfonic Acid (MES), Complexation Reaction of Cu<sup>II</sup>-MES, and Interaction of Hydrous Manganese Oxide Surface with Cu<sup>II</sup> in MES Buffer." CHIMIA 43, no. 1-2 (1989): 13. https://doi.org/10.2533/chimia.1989.13.
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The acidity constants of 2-morpholinoethanesulfonic acid (MES) have been determined at various ionic strengths; pKa1 = 1.99 and pKa2 = 6.21 at ionic strength I = 0.1 M. The conditional stability constant of CuII-MES complex has been measured by means of a CuII-ion selective electrode: lg cKCu-MES = 1.39 ± 0.07 at pH = 5.57. Furthermore the interaction between CuII and hydrous manganese oxide surface in MES buffer was studied by differential pulse polarography. Conditional stability constants for CuII-MnOx complex (SCu) at three pH values were calculated by two methods: van den Berg-Ružić method as well as the Scatchard plot yield corresponding values for lg cKSCu. Both methods indicated the presence of one class of equivalent binding sites. The results of this investigation corroborate that MES is a suitable buffer for studying metal-MnOx interactions.
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Yang, Yu, та Edward P. C. Lai. "Optimization of Molecularly Imprinted Polymer Method for Rapid Screening of 17β-Estradiol in Water by Fluorescence Quenching". International Journal of Analytical Chemistry 2011 (2011): 1–8. http://dx.doi.org/10.1155/2011/214747.
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A new method was optimized for rapid screening of 17β-estradiol (E2) in water under 10 min. Molecularly imprinted polymer (MIP) particles (325 ± 25 nm) were added in a water sample at pH 5.5 and 20∘C to form a suspension. Fluorescence emission from E2 nonspecifically bound onto the MIP particles was first quenched by large gold nanoparticles (43 ± 5 nm). The Stern-Volmer plot was linear, with dynamic quenching constants (Ksv) of 2.9 ×104 M-1. Fluorescence emission from E2 specifically bound inside the MIP particles was next quenched by small nitrite anions that easily penetrated the imprinted cavities. The Stern-Volmer plot became nonlinear, withKsv= 2.1 × 102 M-1and static quenching constant (V) below 1.0 M-1. The difference between these two emission intensities varied as the initial E2 concentration in water, generating a Scatchard calibration curve withR2>0.97from 0.1 to 10 ppb.
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Babbs, Charles F., and David W. Griffin. "Scatchard analysis of methane sulfinic acid production from dimethyl sulfoxide: A method to quantify hydroxyl radical formation in physiologic systems." Free Radical Biology and Medicine 6, no. 5 (1989): 493–503. http://dx.doi.org/10.1016/0891-5849(89)90042-7.
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Singh, Vinod. "Structural requirement for the recognition of luteinizing hormone releasing hormone (LHRH) by monoclonal and conventional anti-LHRH antibodies." Biochemistry and Cell Biology 64, no. 12 (1986): 1372–77. http://dx.doi.org/10.1139/o86-180.
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Immunochemical studies on monoclonal and conventional anti-LHRH antibodies have been reported. The association constants (Ka) were in the order of 109–1010 L/mol when calculated from Scatchard and Steward–Petty plots. Heterogeneity indices (a) calculated from Sips plot were nearly 1.0, indicating the homogeneous nature of monoclonals. Most of the conventional anti-LHRH produced by monkey, baboon, rabbit, and dog, by immunization using LHRH linked to tetanus toxoid by the carbodiimide condensation method, showed a single slope in Scatchard analysis (except two dog antisera) and a values were nearly 1.0. Monoclonals and conventionals reacted most strongly with native LHRH(NH2). Monoclonals showed poor reactivity with LHRH free acid and LHRH fragments containing free acid. The C-terminus tetrapeptide 7–10 showed 10 times more reactivity than tripeptide 4–6. The heptapeptide 4–10 showed 100 and 1000 times more reactivity than the tetra- and tri-peptide, respectively. Introduction of the tripeptide pGlu-His-Trp-OH to heptapeptide 4–10 caused five times more inhibition in reactivity than the heptapeptide. Conventional anti-LHRH antibodies manifested specificity to the C-terminus of LHRH. These sera did not react with LHRH free acid and LHRH fragments of sequence 4–6, 7–10, and 4–10. The complete loss of reactivity of conventional antibodies and poor reactivity of monoclonal antibodies was partly regained when LHRH free acid was coupled to Lys, Lys-MDP, or (Ala)2-tuftsin, suggesting that for monoclonals and conventionals the antigenic determinant was confined to the conformation involving the C-terminus amide of LHRH.
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Rinne, Juha O., Jarmo Hietala, Ulla Ruotsalainen, et al. "Decrease in Human Striatal Dopamine D2 Receptor Density with Age: A PET Study with [11C]Raclopride." Journal of Cerebral Blood Flow & Metabolism 13, no. 2 (1993): 310–14. http://dx.doi.org/10.1038/jcbfm.1993.39.
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The effect of age on human striatal dopamine D2 receptors was investigated with positron emission tomography (PET) using [11C]raclopride as a radioligand. Twenty-one healthy volunteers aged from 20 to 81 years were studied. An equilibrium method was applied and two separate PET scans with different specific activities of [11C]raclopride were performed. The maximal number of receptors ( Bmax) and their dissociation constant ( Kd) were calculated using Scatchard analysis. There was an age-dependent decline in the Bmax ( r = 0.49; p = 0.02) of striatal D2 receptors while the Kd remained unchanged. The results show that there is an age-related loss of striatal D2 receptors, which, together with other changes in the brain nigrostriatal dopaminergic system, may contribute to extrapyramidal symptoms associated with aging.
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Delforge, Jacques, Sabina Pappata, Philippe Millet, et al. "Quantification of Benzodiazepine Receptors in Human Brain Using PET, [11C]Flumazenil, and a Single-Experiment Protocol." Journal of Cerebral Blood Flow & Metabolism 15, no. 2 (1995): 284–300. http://dx.doi.org/10.1038/jcbfm.1995.34.
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A kinetic method using a multiinjection protocol, positron emission tomography (PET), and [11C]flumazenil as a specific ligand was used to study in vivo the flumazenil-benzodiazepine receptor interactions in the human brain. The model structure is composed of three compartments (plasma, free, and bound ligand) and five parameters (including the benzodiazepine receptor concentration). The arterial plasma concentration, after correction for metabolites, was used as the input function. The experimental protocol, which consisted of three injections of labeled and/or unlabeled ligand, allowed the evaluation of the five model parameters in various brain regions from a single experiment. In particular, the concentration of receptor sites available for binding ( B′max) and the equilibrium dissociation constant ( KD VK, VR being the volume of reaction) were estimated in five brain regions, including the pons, in which these parameters are identified for the first time ( B′max = 4.7 ± 1.7 pmol/ml and KD VR = 4.4 ± 1.3 pmol/ml). Due to the large range of measured receptor concentrations, a linear correlation between B′max and KD VR was pointed out ( r = 0.88, p < 0.0005) and was interpreted as a linear relationship between B′max and VR, the parameter KD being assumed constant. This result and its concordance with the published data are discussed. Simulation of the usual two-experiment Scatchard analysis, using the pons as a reference region, showed that the bias on the receptor concentration estimates introduced by this method is significant (from 20 to 40%) but can be corrected using an estimate of the receptor concentration in the pons. Furthermore, we propose a new experimental protocol, based on a Scatchard analysis of the PET data obtained with a partial-saturation experiment. This single-injection protocol is entirely noninvasive, and thus the estimation of the benzodiazepine receptor concentration and of the flumazenil affinity is now possible in human patients using a single 1-h experiment without blood sampling.
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Zarembka, F. R., D. E. Koller, and E. D. Plotka. "Effect of ether or ketamine anesthesia on rat uterine estrogen and progesterone receptors." Clinical Chemistry 35, no. 1 (1989): 143–45. http://dx.doi.org/10.1093/clinchem/35.1.143.
Full textAbstract:
Abstract Rat uterine estrogen receptors (ER) and progesterone receptors (PR) have been used as controls in ER and PR assays of breast tumors. Stunning or decapitation of experimental animals without prior anesthesia is no longer acceptable as a method of killing. Thus, we compared the effects of two anesthetics on the concentration of rat uterine ER and PR. Rats were killed by one of three methods: (a) stunning, (b) ether anesthesia followed by decapitation, or (c) ketamine anesthesia followed by decapitation. ER and PR concentrations were determined by titration assay, with dextran-coated charcoal separation, and quantified by Scatchard analysis. No significant differences were found in mean receptor concentrations or dissociation constants for the three groups. The results indicate that there is no residual effect of diethyl ether or ketamine hydrochloride on the binding of either estrogen or progestin to their respective receptors. The use of decapitation after either ether or ketamine anesthesia is appropriate for measuring ER and PR receptors in rat uteri.
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Katoh, Makoto, Susumu Chishima, Nobukazu Kiuchi, Tomihiro Ikeo, and yasuhiko Sasaki. "A New Method for the Assay of Exposed Platelet Fibrinogen Receptor Using a Chemiluminescent Label." Thrombosis and Haemostasis 74, no. 06 (1995): 1546–50. http://dx.doi.org/10.1055/s-0038-1649980.
Full textAbstract:
SummaryAssay of the platelet fibrinogen-binding receptor glycoprotein (GP) IIb/IIIa is widely performed using 125I-labeled fibrinogen (125I-fibrinogen). We successfully devised a receptor binding assay system with high selectivity and sensitivity using a stable chemiluminescent acridinium derivative I-labeled fibrinogen (acridinium-fibrinogen).Human fibrinogen in saline was labeled with equimolar acridinium dissolved in dimethylformamide, and allowed to react with gel-filtered human platelets in the presence of ADP. Acridinium-fibrinogen binding to GPIIb/IIIa was assayed by measuring chemiluminescence emitted on addition of 0.1 N NaOH containing 0.06% H202 in a luminometer. Non-specific binding was measured in the presence of 10 mM EDTA. Acridinium-fibrinogen binding to human platelets was rapid and reversible, specific and saturable, and dependent on ADP concentrations. Scatchard plot analysis revealed one class of binding sites with a Kd of 326 nM and Bmax of 7.8 pmol/108 platelets. These values were comparable to the data obtained by using 125I-fibrinogen. Unlabeled fibrinogen, RGDS, and HHLGGAKQAGDV (fibrinogen γ-chain 400-411) displaced acridinium-fibrinogen from its binding site with Ki values of 322 nM, 9.2 μM and 31.3μM, respectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotop.
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Ganesan, Sumathi, and Gurumallesh Prabu Halliah. "Synergetic Performance of Graphene Oxide and Chitosan on the Removal of Direct Red 7." Oriental Journal of Chemistry 35, no. 6 (2019): 1789–98. http://dx.doi.org/10.13005/ojc/350623.
Full textAbstract:
Graphene oxide/Chitosan (GOCH) composite was synthesized by hydrothermal method and structurally characterized by FT-IR, RAMAN, XRD and BET analyses which provide support for graphene oxide and chitosan incorporation. The synthesized composite was employed for the removal of direct red 7 (DR7) by batch adsorption process. Langmuir, Freundlich, Temkin, Dubinin-Radushkevic, Harkin-Jura, Scatchard plot analysis and Hasley isotherms were used to elucidate adsorption mechanism. The value of R2 revealed that isotherm was well explained by Langmuir model. The extent of monolayer adsorption capacity of GOCH was calculated as 34.2 mg/g. The pseudo first order kinetic studies were in agreement with experimental data. Thermodynamic parameters such as activation energy (Ea = 8.405 KJ/mol), enthalpy (ΔH = 89.417 KJ/mol), free energy change (ΔG) and entropy (ΔS = 0.2971 KJ/mol) were calculated. It propounded that the adsorption of DR7 on GOCH was favorable, spontaneous and an endothermic process.
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Royani, Idha, Widayani, Abdullah Mikrajuddin, and Khairurrijal. "Effect of Heating Time on Atrazine-Based MIP Materials Synthesized via the Cooling-Heating Method." Advanced Materials Research 896 (February 2014): 89–94. http://dx.doi.org/10.4028/www.scientific.net/amr.896.89.
Full textAbstract:
Molecular imprinting is a technique to produce a polymer called as molecularly imprinted polymer (MIP) that provides cavities to form a particular space generated by removing the template when the polymer has been formed. It will recognize a target that has the shape and physico-chemical properties similar or identical with those of template molecule. In this study, MIPs using atrazine as template have been made via the cooling-heating method. Initially the pre-polymer solution was cooled at a refrigerator for 1 h. Next, the polymerization was carried out at 70 °C for heating times of 90, 120, and 150 min. without nitrogen flow which is generally done for polymerization process. Characterizations were performed by employing a reversed-phase high performance liquid chromatograph (HPLC) and scanning electron microscope (SEM). From Scatchard plots, it was found that the equilibrium dissociation constantKDand the apparent maximum number of binding sitesBmax, which are written as (KD,Bmax), are (4.69 μM, 9.87 mmol/g), (4.54 μM, 9.56 mmol/g) and (3.52 μM, 7.44 mmol/g) for the heating times of 150, 120, and 90 min., respectively. This is verified by their SEM images showing that the broadest pore size distribution with the highest number of pores is in the MIP prepared under the heating time of 150 min. The MIPs therefore could be applied as an atrazine sensor and the MIP prepared under the heating time of 150 min. would give its best characteristics compared to the others.
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Luo, Yong Ming, Yong Dong Luo, Fang You Chen, Xue Lian Zhang, and Xiao Ying Yin. "Preparation of Henriol C Molecularly Imprinted Polymer Microspheres." Applied Mechanics and Materials 664 (October 2014): 34–37. http://dx.doi.org/10.4028/www.scientific.net/amm.664.34.
Full textAbstract:
Molecular imprinted polymer micrcosheres (MIPMs) were prepared through precipitation polymerization by henriol C using as the template molecule. The morphologies of synthesized MIPMs were characterized by scanning electronmicroscope (SEM). Systematic investigations of the influences of key synthetic conditions, including functional monomers, cross-linkers and porogens, on the morphologies, yields and the recognition properties of the MIPs were conducted. The results indicated that the morphologies of MIPs with DVB as cross-linker was perfect, but their binding affinity is lower than that of MIPs with TRIM or EDMA as cross-linkers. And particle size of MIPs with TRIM as cross-linkers is small but with high binding affinity. Scatchard analysis revealed that the homogeneous binding sites were formed in the polymers. The application of MIPs with high affinity and excellent stereo-selectivity toward henriol C in solid-phase extraction (SPE) column might offer a novel method for the enrichment and determination of sesquiterpenoids in the traditional herbal medicine.
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Yu, Jin Yang, Xiao Ling Hu, Ren Yuan Song, and Shan Xi. "Molecularly Imprinted Polymer Microspheres Prepared by Precipitation Polymerization for Atenolol Recognition." Advanced Materials Research 148-149 (October 2010): 1192–98. http://dx.doi.org/10.4028/www.scientific.net/amr.148-149.1192.
Full textAbstract:
Molecularly imprinted polymer microspheres for selective binding and recognition of atenolol were prepared by means of precipitation polymerization method using methacylic acid as functional monomer and trimethylolpropane trimethacrylate as cross-linker in the presence of atenolol as template molecule in acetonitrile solution. Computer simulation was employed to demonstrate the mechanism of the interaction between methacylic acid and atenolol. The scanning electron microscopy exhibited that the polymers were uniform spheres with the diameter of about 0.6µm. The adsorption properties of atenolol for imprinted microspheres were evaluated by equilibrium rebinding experiments. Scatchard plot analysis revealed that there were two classes of binding sites in the imprinted microspheres. The dissociation constant and the apparent maximum binding capacity were 4.56×10-4mol/L and 186.46μmol/g for the high affinity binding sites, 2.40×10-2mol/L and 4.01mmol/g for the low affinity binding sites. Compared to the structrally analogues, the imprinted microspheres exhibited a high selective reconizable capacity towards the template.
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Yin, Xiao Ying, Liu Qing Shan, Xiu Lin Han, and Yong Ming Luo. "Preparation and Recognition Properties of Andrographolide Molecularly Imprinted Ploymer Microspheres." Advanced Materials Research 160-162 (November 2010): 777–82. http://dx.doi.org/10.4028/www.scientific.net/amr.160-162.777.
Full textAbstract:
Molecular imprinted polymer micrcosheres (MIPMs) were prepared through precipitation polymerization by andrographolide using as the template molecule. The morphologies of synthesized MIPMs were characterized by scanning electronmicroscope (SEM). Systematic investigations of the influences of key synthetic conditions, including functional monomers, cross-linkers and porogens, on the morphologies, yields and the recognition properties of the MIPs were conducted. The results indicated that the morphologies of MIPs with DVB as cross-linker was perfect, but their binding affinity is lower than that of MIPs with TRIM or EDMA as cross-linkers. And particle size of MIPs with TRIM as cross-linkers is small but with high binding affinity. Scatchard analysis revealed that the homogeneous binding sites were formed in the polymers. The application of MIPs with high affinity and excellent stereo-selectivity toward andrographolide in solid-phase extraction (SPE) column might offer a novel method for the enrichment and determination of terpenoids compounds in the traditional herbal medicine.
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Dittadi, R., M. Gion, A. Brazzale, and G. Bruscagnin. "Radioligand binding assay of epidermal growth factor receptor: causes of variability and standardization of the assay." Clinical Chemistry 36, no. 6 (1990): 849–54. http://dx.doi.org/10.1093/clinchem/36.6.849.
Full textAbstract:
Abstract Although experimental evidence indicates a probable role of epidermal growth factor receptor (EGFr) in clinical oncology, no standardized method for its determination has been yet described, and discrepant results have been reported in clinical studies. In standardizing a radioligand binding assay for EGFr, we evaluated the causes of variability in each step of the assay. Entrapment of EGFr in the nuclear fraction and contamination of the crude membrane fraction by cytosol protein were eliminated through preliminary purification steps. Both Scatchard and Rosenthal analysis of the saturation reaction of the membrane fraction with a wide range of concentrations of 125I-labeled EGF revealed a double class of binding sites. Study of the saturation reaction showed a partial exchange of 125I-labeled EGF with endogenous EGF within 20 h. The present method--incubation of partly purified membrane fraction with 125I-labeled EGF, 0.5 nmol/L, with and without 100-fold excess of cold EGF, for 20 h at 26 degrees C, followed by centrifugation at 5000 x g for 30 min to separate membrane-bound 125I-labeled EGF--shows good sensitivity, precision, and accuracy; is reasonably simple; and may be suitable for routine clinical use.
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D., B. Patil, D. Patil V., R. Pujari S., and B. Deshmukh M. "Spectroscopic studies of molecular interactions involving aniline and substituted aniline donors and chloranil as an electron acceptor in a binary solvent mixtures." Journal of Indian Chemical Society Vol. 82, Jun 2005 (2005): 530–33. https://doi.org/10.5281/zenodo.5830600.
Full textAbstract:
Chemistry Department, Vivekanand College, Kolhapur-416 003, India TKIET, Warananagar, Dist. Kolhapur, India Department of Chemistry, Shivaji University, Kolhapur-416 004, India <em>Manuscript received 13 January 2004, revised 1 February 2005, accepted 9 March 2005</em> Charge-transfer complexes of chloranil acceptor with aniline and substituted anilines as donors were studied in binary solvent mixtures of different composition of benzene and dichloromethane (by volume) at room temperature. The stoichiometry of the complexes was established to be 1 : 1. For this purpose, optical data were subjected to the form of the Rose-Drago equation for 1 : 1 equilibria. The stoichiometry was also tested by other methods such as Scott, Scatchard in addition to Bensi-Hildebrand method. The formation constant (<em>k</em><sub>c</sub>) and molar absorptivities (ϵ<sub>λ</sub>) of complexes were determined by least square method. Linear relationship was found between oscillator strength (f) and refractive index function <em>n</em><sup>2</sup>-1/ 2<em>n</em><sup>2</sup> + 1. The values of <em>hv</em><sub>CT</sub> were correlated with ionization potential of the donor and a linear relationship was obtained with a slope approximating to unity allowing the determination of the energy of interaction in the excited state. Chargetransfer (CT) band maxima of complexes show red shifts in passing from primary→ secondary→ tertiary a mines.
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Krambovitis, E., G. Hatzidakis, A. Hatzoglou, et al. "Estrogen and progesterone receptors in breast cancer microsamples simultaneously quantified by enzyme-ligand immunoassay." Clinical Chemistry 41, no. 1 (1995): 48–53. http://dx.doi.org/10.1093/clinchem/41.1.48.
Full textAbstract:
Abstract A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).
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Rahmani, Mahdiyeh Ebrahimi, Mehdi Ansari, Mohammadreza Nateghi, and Maryam Kazemipour. "Computation-Assisted Molecularly Imprinted Polymer Synthesis for Extraction of Naltrexone from Urine Using Experimental Design and Determination by UPLC–DAD." Journal of AOAC INTERNATIONAL 100, no. 3 (2017): 700–711. http://dx.doi.org/10.5740/jaoacint.16-0226.
Full textAbstract:
Abstract To design a molecularly imprinted polymer (MIP) for naltrexone, calculations were performed using Gaussian 03 software,and the interaction energy (ΔE) of template–monomer complexes was estimated using the density functional theory method with the B3LYP function and 6-311G (d) basis set. The effect of different solvents in the polymerization process was studied using the polarizable continuum model. It was shown that five molecules of methacrylic acid gave the largestΔE with tetrahydrofuran as the polymerization solvent.Effective factors of the removal efficiency of naltrexone by the MIP were selected using a central composite design, and thereafter, the optimization of significant factors was performed by response surface methodology. The results predicted through these models showed good agreement with experimental values. The adsorption amount, selectivity distribution coefficient, and selectivity coefficient were found to be 11.60 mg/g, 35.31, and 2.27, respectively. Experiments of naltrexone adsorption onto the MIP were in accordance with the first-orderand Langmuir-Freundlich adsorption models. By applying the data to the Scatchard equation, the KD and Qmax were determined as 526.31 mg/L and 19.47 mg/g, respectively.
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Galo, André Luiz, and Márcio Francisco Colombo. "Singular Value Decomposition and Ligand Binding Analysis." Journal of Spectroscopy 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/372596.
Full textAbstract:
Singular values decomposition (SVD) is one of the most important computations in linear algebra because of its vast application for data analysis. It is particularly useful for resolving problems involving least-squares minimization, the determination of matrix rank, and the solution of certain problems involving Euclidean norms. Such problems arise in the spectral analysis of ligand binding to macromolecule. Here, we present a spectral data analysis method using SVD (SVD analysis) and nonlinear fitting to determine the binding characteristics of intercalating drugs to DNA. This methodology reduces noise and identifies distinct spectral species similar to traditional principal component analysis as well as fitting nonlinear binding parameters. We applied SVD analysis to investigate the interaction of actinomycin D and daunomycin with native DNA. This methodology does not require prior knowledge of ligand molar extinction coefficients (free and bound), which potentially limits binding analysis. Data are acquired simply by reconstructing the experimental data and by adjusting the product of deconvoluted matrices and the matrix of model coefficients determined by the Scatchard and McGee and von Hippel equation.
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Nosjean, Olivier, Sophie Souchaud, Clarisse Deniau, Olivier Geneste, Nicolas Cauquil, and Jean A. Boutin. "A Simple Theoretical Model for Fluorescence Polarization Binding Assay Development." Journal of Biomolecular Screening 11, no. 8 (2006): 949–58. http://dx.doi.org/10.1177/1087057106294841.
Full textAbstract:
Fluorescence polarization is a screening technology that is radioactivity free, homogeneous, and ratiometric. The signal measured with this technology is a weighted value of free and bound ligand. As a consequence, saturation curves are accessible only after calculation of the corresponding concentrations of free and bound ligand. To make this technology more accessible to assay development, the authors propose a simple mathematical model that predicts fluorescence polarization values from ligand and receptor total concentrations, depending on the corresponding dissociation constant. This model was validated using data of Bodipy-NDP-αMSH binding to MC5, obtained after either ligand saturation of a receptor preparation or, conversely, receptor saturation of a ligand solution. These experimental data were also used to calculate the actual concentration of free and bound ligand and receptor and to obtain pharmacological constants by Scatchard analysis. A general method is proposed, which facilitates the design of fluorescence polarization binding assays by relying on the representation of theoretical polarization values. This approach is illustrated by the application to 2 systems of very different affinities.
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Delforge, J., L. Spelle, B. Bendriem, Y. Samson, and A. Syrota. "Parametric Images of Benzodiazepine Receptor Concentration Using a Partial-Saturation Injection." Journal of Cerebral Blood Flow & Metabolism 17, no. 3 (1997): 343–55. http://dx.doi.org/10.1097/00004647-199703000-00011.
Full textAbstract:
The in vivo quantification of the benzodiazepine receptor concentration in human brain using positron emission tomography (PET) and 11C-flumazenil (11C-FMZ), is usually based on a three-compartment model and on PET curves measured in a small number of large regions of interest; however, it should be interesting to estimate the receptor concentration for each pixel and to build quantified images of the receptor concentration. The main advantage is to allow screening of the receptor site localization and visual observation of the possible abnormalities. Up to now, all the methods described include complex experimental protocols, difficult to use in routine examinations. In this paper, we propose the partial-saturation approach to obtain parametric images of benzodiazepine receptor concentration and FMZ affinity. It consists of a single FMZ injection with a low specific activity, followed by Scatchard analysis. Like other parametric imaging methods, this partial-saturation approach can lead to a small percentage (<1%) of unrealistic values in receptor-poor regions; however, it is the only method that allows receptor concentration and affinity images to be obtained from a single-injection 40-min experiment without blood sampling. We also propose a second method in which the receptor concentration map is directly deduced from the PET image acquired 5 to 10 min after a partial-saturation injection. This method assumes a known and constant FMZ affinity value but requires only very simple corrections of this PET image. It is robust (negative values are never found) and quite simple to use in routine examination of patients (no blood sampling, single injection, only 10-min experiment).
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Farhadi, K., R. Tahmasebi, P. Biparva, and R. Maleki. "In vitro study of the binding between chlorpyrfos and sex hormones using headspace solid-phase microextraction combined with high-performance liquid chromatography." Human & Experimental Toxicology 34, no. 8 (2015): 819–27. http://dx.doi.org/10.1177/0960327114559990.
Full textAbstract:
Endocrine-disrupting chemicals are compounds that alter the normal functioning of the endocrine system. Organophosphorus insecticides, as chlorpyrifos (CPS), receive an increasing consideration as potential endocrine disrupters. Physiological estrogens, including estrone (E1), 17β-estradiol (E2), and diethylstilbestrol (DES) fluctuate with life stage, suggesting specific roles for them in biological and disease processes. There has been great interest in whether certain organophosphorus pesticides can affect the risk of breast cancer. An understanding of the interaction processes is the key to describe the fate of CPS in biological media. The objectives of this study were to evaluate total, bound, and freely dissolved amount of CPS in the presence of three estrogenic sex hormones (ESHs). In vitro experiments were conducted utilizing a headspace solid phase microextraction (HS-SPME) combined with high-performance liquid chromatography (HPLC) method. The obtained Scatchard plot based on the proposed SPME-HPLC method was employed to determine CPS-ESHs binding constant and the number of binding sites as well as binding percentage of each hormone to CPS. The number of binding sites per studied hormone molecule was 1.10, 1, and 0.81 for E1, E2, and DES, respectively. The obtained results confirmed that CPS bound to one class of binding sites on sex hormones.
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Wang, Caihua, Changhao Li, Haibing Zhou, and Jian Huang. "High-Throughput Screening Assays for Estrogen Receptor by Using Coumestrol, a Natural Fluorescence Compound." Journal of Biomolecular Screening 19, no. 2 (2013): 253–58. http://dx.doi.org/10.1177/1087057113502673.
Full textAbstract:
Estrogen receptor (ER) is a ligand-inducible transcriptional factor involving in cell growth, differentiation, and diseases, so detection and identification of compounds having estrogenic effects are of great importance in the drug discovery industry. We have developed and validated a rapid, simple, and homogeneous method that can detect estrogenic compounds. This human ERα/β binding assay uses fluorescence polarization (FP) by applying an autofluorescent phytoestrogen, coumestrol (CS). A nonspecific adsorption assay shows that no obvious nonspecific adsorption is detected between CS and ERs. In the Scatchard plot analysis, the convex curve exhibits a positive cooperative binding, indicating that the binding of CS induces a conformational change of the ER to form a dimer and increases the affinity for the additional CS. In the Hill plot analysis, CS shows moderate binding affinity with both ERα and ERβ, and the measured Kd of CS is 32.66 µM and 36.14 µM, respectively, indicating that CS is applicable to the ER binding assay for determination of potent ligands of moderate binding affinity. Four typical ligands are selected to verify the ER binding assays, and the results are consistent with the reported data. All of above make the FP method based on CS suitable for high-throughput screening.
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Bolufer, P., E. Ricart, A. Lluch, et al. "Aromatase activity and estradiol in human breast cancer: its relationship to estradiol and epidermal growth factor receptors and to tumor-node-metastasis staging." Journal of Clinical Oncology 10, no. 3 (1992): 438–46. http://dx.doi.org/10.1200/jco.1992.10.3.438.
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PURPOSE The present report attempts to clarify whether there is a relationship between aromatase activity (ARAC) and estradiol (E2), hormonal receptors, E2 receptor (ER), and epidermal growth factor receptor (EGFR), as well as with tumor stage and histopathology in human breast cancers. MATERIALS AND METHODS We studied 225 breast carcinomas, 67 of which were premenopausal and 158 postmenopausal. In each sample, ARAC, EGFR, ER, and E2 were quantified. ARAC was quantified by Thompson and Siiterii's method, EGFR was quantified with a two-point assay method using radioactive iodine (125I)-EGF as ligand, and ER was measured by the Scatchard method using 3H-E2. E2 was quantified by radioimmunoassay in the diethylether tumor extract. RESULTS ARAC was found in 64% of the cancers studied. There is a strong direct association between ARAC and tumor size in postmenopausal patients (P = .001). In the postmenopausal group, the proportion of ARAC-positive (ARAC+) tumors is significantly higher among ER-positive (ER+) than ER-negative (ER-) ones (P less than .001). ER+ tumors also have significantly higher levels of E2 than do ER- ones (P less than .0001); similarly, ARAC+ tumors have significantly higher levels of E2 than do ARAC- ones (P less than .0001). There is a significant multiple linear correlation between the log of the levels of ARAC, ER, and EGFR and the log of tumor E2 (P less than .0001). The correlation coefficients obtained show that ARAC and ER have a positive effect on tumor E2. CONCLUSION The results obtained suggest the importance of tumor ARAC in the tumoral levels of E2 and reinforce the possible biologic significance of tumor ARAC, especially in postmenopausal breast carcinoma patients.
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Grimditch, G. K., R. J. Barnard, S. A. Kaplan, and E. Sternlicht. "Insulin binding and glucose transport in rat skeletal muscle sarcolemmal vesicles." American Journal of Physiology-Endocrinology and Metabolism 249, no. 4 (1985): E398—E408. http://dx.doi.org/10.1152/ajpendo.1985.249.4.e398.
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A new method is described for isolation of sarcolemma (SL) from skeletal muscle of rats that produces vesicles of high purity and yield. There was a mean 59-fold purification (n = 22) of the SL marker enzyme K+-p-nitrophenylphosphatase. Specific activities of marker enzymes for sarcoplasmic reticulum and mitochondria were low, indicating minimal contamination. Despite the high purity and low contamination, a relatively high protein yield was achieved (0.43 +/- 0.03 mg/g wet wt, n = 25). Electron microscopy showed that the membranes were primarily vesicles. Specific 125I-insulin binding association constants derived from the high- and low-affinity portion of the Scatchard plots were 0.764 +/- 0.154 and 0.0096 +/- 0.0012 X 10(9) M-1, whereas the apparent number of receptors were 15.0 +/- 4.1 and 925 +/- 80 X 10(9) per mg of SL protein. Equilibrium exchange glucose transport studies at 37 degrees C indicated that the SL vesicles exhibited specific D-glucose transport which was responsive to in vivo insulin stimulation. We conclude that this isolation procedure, especially in light of the high purity and yield, provides a good and practical experimental model for studying insulin binding and glucose transport in skeletal muscle.
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Jin, Ya-Feng, Yi-Jun Zhang, Yu-Ping Zhang, Jun Chen, Xiao-Mao Zhou, and Lian-Yang Bai. "Synthesis and Evaluation of Molecularly Imprinted Polymer for the Determination of the Phthalate Esters in the Bottled Beverages by HPLC." Journal of Chemistry 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/903210.
Full textAbstract:
A molecularly imprinted polymer (MIP) was prepared in acetonitrile by bulk polymerization, using di-n-octylphthalate (DOP) as a template molecular,α-methacrylic acid (MAA) as a functional monomer, and ethylene dimethacrylate (EDMA) as a crosslinker. Characterization and evaluation of the prepared MIP were carried out by scanning electron microscope (SEM), infrared absorption spectroscopy (IR), and the Scatchard analysis, respectively. Through the optimization of washing solvent, eluting solvent amount, flow rate of loading solution, and loading sample volume, an analysis method was established for DOP related compounds with high selectivity and sensitivity by using the selective molecularly imprinted solid-phase extraction (MI-SPE) technique. Moreover, under the optimal conditions, the extraction effects were comparatively investigated by using MIP cartridge, NIP cartridge, and the commercial PLS cartridge used especially for phthalic acid esters (PAEs), respectively. The results showed that the recoveries of spiked PAEs are in the range of 90.4%–97.8% with the relative standard deviation (RSD) of 1.6%–3.8% on the resulted MIP cartridge, whilst lower recoveries were obtained ranging from 80.2% to 88.9% with an RSD of 1.4%–5.2% on the commercial PLS cartridge.
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Zhang, Li, Hongbo Mo, Chuan Wang, et al. "Synthesis and Properties of Cefixime Core–Shell Magnetic Nano-Molecularly Imprinted Materials." Polymers 15, no. 22 (2023): 4464. http://dx.doi.org/10.3390/polym15224464.
Full textAbstract:
Novel core–shell magnetic molecularly imprinted polymers (MMIPs) were synthesized using the sol–gel method for the adsorption of cefixime (CFX). Fe3O4@SiO2 is the core, and molecularly imprinted polymers (MIPs) are the shell, which can selectively interact with CFX. The preparation conditions, adsorption kinetics, adsorption isotherms, selective adsorption ability, and reutilization performance of the MMIPs were investigated. The adsorption capacity of MMIPs for CFX was 111.38 mg/g, which was about 3.5 times that of MNIPs. The adsorption equilibrium time was 180 min. The dynamic adsorption experiments showed that the adsorption process of MMIPs to CFX conformed to the pseudo-second-order model. Through static adsorption study, the Scatchard analysis showed that MMIPs had two types of binding sites—the high-affinity binding sites and the low-affinity binding sites—while the Langmuir model fit the adsorption isotherms well (R2 = 0.9962). Cefepime and ceftiofur were selected as the structural analogs of CFX for selective adsorption studies; the adsorption of CFX by MMIPs was higher than that of other structural analogs; and the imprinting factors of CFX, cefepime, and ceftiofur were 3.5, 1.7, and 1.4, respectively. Furthermore, the MMIPs also showed excellent reusable performance.
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Christiansen, K., and J. Carlsen. "Insertion of isolated insulin receptors into placental membrane vesicles." Biochemical Journal 281, no. 2 (1992): 425–30. http://dx.doi.org/10.1042/bj2810425.
Full textAbstract:
Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.
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van Zoelen, E. J. J. "Receptor-ligand interaction: a new method for determining binding parameters without a priori assumptions on non-specific binding." Biochemical Journal 262, no. 2 (1989): 549–56. http://dx.doi.org/10.1042/bj2620549.
Full textAbstract:
Analysis of receptor-ligand binding characteristics can be greatly hampered by the presence of non-specific binding, defined as low-affinity binding to non-receptor domains which is not saturable within the range of ligand concentrations used. Conventional binding analyses, e.g. according to the methods described by Scatchard or Klotz, relate the amount of specific receptor-ligand binding to the concentration of free ligand, and therefore require assumptions on the amount of non-specific binding. In this paper a method is described for determining the parameters of specific receptor-ligand interaction which does not require any assumption or separate determination of the amount of non-specific binding. If the concentration of labelled free ligand is constant, a plot of Fu/(B0*-B*) versus Fu yields a linear relationship, in the case of a single receptor class, in which Fu is the concentration of unlabelled free ligand, B0* is the total amount of labelled bound ligand in the absence of unlabelled ligand and B* is the total amount of labelled bound ligand in the presence of an unlabelled ligand concentration Fu; all of these data are readily obtained from binding studies. This linear relationship holds irrespective of the amount of non-specific binding, and the values for receptor density, ligand dissociation constant and a constant for non-specific binding can be readily obtained from it. If the concentration of labelled free ligand is not a constant for all data points, data are first converted according to a straightforward normalization procedure to permit the use of this relationship. The presence of multiple receptor classes with dissociation constants in the range of the ligand concentrations used results in a negative deviation from this linearity, and therefore the presence of multiple receptor classes can be discriminated unequivocally from non-specific binding. Both theoretical and practical advantages of the present method are described. The method, which will be referred to as the linear subtraction method, is illustrated using the binding of tumour promoters and polypeptide growth factors to their specific cellular receptors.
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Tsuchida, J., S. Ueki, Y. Takada, Y. Saito, and J. Takagi. "The ‘ligand-induced conformational change’ of alpha 5 beta 1 integrin. Relocation of alpha 5 subunit to uncover the beta 1 stalk region." Journal of Cell Science 111, no. 12 (1998): 1759–66. http://dx.doi.org/10.1242/jcs.111.12.1759.
Full textAbstract:
Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-beta1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of beta1 subunits. The binding of a 125I-labeled AG89 Fab fragment to alpha5 beta1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated alpha5 beta1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only beta1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional beta1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant beta1 that cannot complex with alpha5. These data suggest that the AG89 epitope is masked by the alpha5 subunit. Ligand binding and integrin activation may uncover the beta1 stalk region by triggering a conformational shift of alpha5 relative to beta1.
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Taylor, J. A., D. N. Salter, A. I. Montgomery, and B. Seve. "Adipocyte Beta-Adrenoceptor numbers in growing pigs of different genotypes." Proceedings of the British Society of Animal Production (1972) 1991 (March 1991): 139. http://dx.doi.org/10.1017/s0308229600020894.
Full textAbstract:
Catecholamines are the major lipolytic agents in fat cells and are mediated primarily by beta-adrenergic receptors. The present experiment was designed to study p-receptor numbers in growing pigs of various genotypes in relation to their body compositions.Fat samples from 5 representative pigs of each genotype were removed immediately after slaughter, at about 60 kg body weight, and stored at -80°C prior to analysis. Adipocytes were extracted by incubation with 1 mg/g (w/v) collagenase in a tris/MgCl2/ EDTA/Bovine serum albumin buffer (pH 7.4) for 1 hour at 37°C. Cells were mechanically ruptured and membranes were removed by ultracentrifugatlon (37,000 rpm for 20 minutes). Recovery was estimated by protein assay using the method of Lowry (1951). Approximately 20 μg of adipocyte membrane protein in 300 μl tris/MgCl2/EDTA assay buffer was added to graded concentrations of the 125I-labelled β-antagonist Iodocyanopindolol (100 μl) together with 100 μl of either assay buffer or the non-specific β-antagonist propanolol (to determine non-specific binding). Samples were then incubated for 1 hour at 37°C. Total β-receptor numbers (maximum binding - Bmax) expressed as pmol/g protein, were estimated by Scatchard analysis using the computer programme ‘EBDA’ by Elsevier - BIOSOFT, Cambridge.