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Affinity Constant (affinity + constant)
Selected AbstractsEnantiomeric separation of aminoglutethimide by capillary electrophoresis using native cyclodextrins in single and dual systemsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2003Adel M. Abushoffa Abstract Aminoglutethimide (AGT) is one of the few examples of chiral drugs that can be enantioseparated by capillary electrophoresis using any of the three native cyclodextrins: ,-, ,-, or ,-CD. A complete resolution of the enantiomers of this compound in cationic form could be achieved with each of the three CDs, using a pH 3 phosphoric acid-triethanolamine buffer. Affinity constants for AGT enantiomers with the three native CDs were determined, confirming that the highest selectivity was given by ,-CD while the strongest complexation was obtained with ,-CD. However, an opposite affinity pattern was observed with the latter. Selectivity was lower for AGT enantiomers in dual CD systems, compared to that obtained with a single selector at its optimal concentration, which confirms that dual systems are of more limited interest when the two selectors have a similar effect on the analyte mobility. These results are in good agreement with those predicted using recently developed mathematical models. [source] Voltammetric Studies of the Interactions Between Ferrocene-Labeled Glutathione and Proteins in Solution or Immobilized onto SurfaceELECTROANALYSIS, Issue 16 2009Yong Peng Abstract Glutathione (GSH) tagged with a ferrocene (Fc) label at its C-terminal was synthesized via coupling ferrocenyl amine to glutathione using o -(benzotriazol-1-yl)- N,N,N,,N, -tetramethyluronium (HBTU)/1-hydroxybenzotrizole (HOBt). The presence of Fc yielded well defined voltammetric signals, rendering the Fc-tagged GSH (GSH-Fc) suitable for electrochemical studies of GSH binding to other biological species. The interaction of GSH-Fc with bovine serum albumin (BSA) was investigated, and a binding ratio of 1.41±0.06 (GSH-Fc/BSA) and an affinity constant Ka of 6.53±2.01×106,M,1 were determined. These results compare well with those measured by fluorescence using untagged GSH, suggesting that the attachment of Fc to GSH does not significantly perturb the GSH structure and binding behavior. By contrasting the binding behavior to several compounds that are known to conjugate to different domains of BSA, the voltammetric study confirmed that GSH-Fc binds at subdomain IIA of BSA with high affinity. The versatility of GSH-Fc for studying GSH binding to surface-confined proteins was also demonstrated with the GSH binding to electroinactive Zn-metallothionein (Zn7 -MT) through hydrogen binding at the region between the Zn7 -MT , and , domains. [source] Assessment of CD8 involvement in T,cell clone avidity by direct measurement of HLA-A2/Mage3 complex density using a high-affinity TCR like monoclonal antibodyEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2005Karine Bernardeau Abstract Peptide affinity for MHC molecules determines the number of MHC/peptide complexes stabilized at the cell surface in in vitro tests or in vaccination protocols. We isolated a high affinity monoclonal antibody specific for the HLA-A2/Mage3 complex that enables an equilibrium binding assay to be performed on T2 cell line loaded with a range of Mage3 peptides. Binding of Mage3 to the HLA-A2 molecule can be modeled by a standard receptor-ligand interaction characterized by an affinity constant. This model enables the measurement of the affinity of other immunogenic peptides for HLA-A2 by a competition test and the calculation of the density of complexes stabilized at the T2 cell surface for all peptide concentrations. Quantification of the HLA-A2/Mage3 complexes at target cell surfaces was used to estimate the number of complexes required to reach cytotoxicity ED50 of human T,cell clones sorted from an unprimed repertoire. We confirm with this antibody the direct relationship between clone avidity and TCR affinity, and the moderate contribution of the CD8 co-receptor in the reinforcement of TCR-MHC/peptide contact. Nevertheless, CD8 plays a critical role in the amplification of the specific signal to establish an efficient T,cell response at low specific complex densities found in physiological situations. [source] High epitope density in a single protein molecule significantly enhances antigenicity as well as immunogenicity: a novel strategy for modern vaccine development and a preliminary investigation about B,cell discrimination of monomeric proteinsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2005Wanli Liu Abstract Although early studies have shown a close correlation between epitope density and epitope-specific humoral immune responses, few attempts have been made to quantitatively compare the antigenic and immunogenic differences between protein molecules bearing low or high degrees of epitope density, nor have studies quantitatively investigated the mechanism of B,cell discrimination of monomeric antigens. In this study, we prepared glutathione S-transferase (GST) fusion proteins bearing various copies of the M2e epitope from the influenza virus M2,protein [GST-(M2e)8, GST-(M2e)4 and GST-(M2e)1], which were used to detect and compare the real-time kinetic binding with M2e-specific mAb by surface plasma resonance. Our data show clearly that fusion proteins bearing higher M2e epitope density resulted in higher average avidity for M2e-specific mAb. Furthermore, it was observed that fusion proteins bearing high M2e epitope density could induce polyclonal antibodies (pAb) with enhanced an average affinity constant (KA) for M2e epitope peptide compared to fusion proteins bearing low epitope density. The average KA of pAb induced by GST-(M2e)8 (3.08 × 108,M,1 or 9.96 × 108,M,1) was up to two orders of magnitude greater than the average KA of pAb induced by GST-(M2e)1 (2.00 × 106,M,1 or 3.43 × 106,M,1). Thus, the data presented here demonstrate that high epitope density in a single protein molecule significantly enhances antigenicity and immunogenicity. These findings enrich our knowledge of how epitope density might relate to the recognition, activation and antibody production processes of epitope-specific immature B,cells. [source] Sorption of tannic acid on zirconium pillared clayJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 1 2002P Vinod Abstract Zirconium pillared clay (PILC) was prepared using montmorillonite as the base clay. Adsorption of tannic acid (tannin) was studied by a batch equilibrium technique, as a function of adsorbate concentration, temperature, pH, agitation speed, particle size of the adsorbent and ionic strength. The process of uptake is governed by diffusion controlled first-order reversible rate kinetics. The higher uptake for the pH range 4.0,6.0 was attributed to external hydrogen bonding between phenolic-OH groups of tannin molecules and the hydrogen bonding sites on the clay. The removal of tannin by adsorption was found to be >99.0% depending on the initial concentration in the pH range of 4.0,6.0. The process involves both film and pore diffusion to different extents. The effects of solute concentration, temperature, agitation speed and particle size on the diffusion rate were investigated. Tannin uptake was found to increase with ionic strength due to the compression of diffuse double layers. The applicability of Langmuir and Freundlich isotherm models has been tested. The maximum adsorption capacity of PILC was found to be 45.8,µmol,g,1 of clay and the affinity constant is 2.9,×,10,2,dm3,µmol,1 at 30,°C. Thermodynamic parameters such as ,G,°,,H,° and ,S,° were calculated to predict the nature of adsorption. The isosteric enthalpies of adsorption were also determined and found to decrease with increasing surface coverage. Regeneration with hot water (60,°C) has been investigated for several cycles with a view to recovering the adsorbed tannin and also restoring the sorbent to its original state. Copyright © 2001 Society of Chemical Industry [source] Evaluation of a new automated enzyme fluoroimmunoassay using recombinant plasmid dsDNA for the detection of anti-dsDNA antibodies in SLEJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2002D. Villalta Abstract ELISA methods to detect anti-double-stranded DNA (anti-dsDNA) antibodies are highly sensitive, but are less specific for the diagnosis of SLE than the immunofluorescence test on Crithidia luciliae (CLIFT) and the Farr assay because they also detect low-avidity antibodies. This study evaluated the specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of a new automated fluoroimmunoassay (EliA dsDNA; Pharmacia, Freiburg, Germany). We compared the results with those obtained using a commercial CLIFT and an in-house anti-dsDNA IgG ELISA method, and verified its putative ability to detect only high-avidity anti-dsDNA antibodies. Sera from 100 SLE patients and 120 controls were studied. The control group included 20 healthy donors, 70 patients with other rheumatic diseases (32 systemic sclerosis (SSc); 18 primary Sjögren syndrome (pSS), 20 rheumatoid arthritis (RA)), and 30 patients with various infectious diseases (ID). Anti-dsDNA avidity was estimated using an ELISA method based upon the law of mass action, and a simplified Scatchard plot analysis for data elaboration; the apparent affinity constant (Kaa) was calculated and expressed as arbitrary units (L/U). Sensitivity, specificity, PPV, and NPV for SLE were 64%, 95.8%, 93.8% and 72.7%, respectively, for the EliA anti-dsDNA assay; 55%, 99.2%, 98.5%, and 68.8%, respectively, for the CLIFT; and 64%, 93.3%, 90.6%, and 72.3%, respectively, for the in-house ELISA. Although EliA anti-dsDNA was positive mainly in SLE patients with high- (Kaa>80 L/U) and intermediate- (Kaa 30,80 L/U) avidity antibodies (45.3% and 49.9%, respectively), it was also positive in five (7.8%) SLE patients with low-avidity anti-dsDNA antibodies, and five controls (three SSc, one pSS, and one ID) (mean Kaa = 16.4 ± 9.04 L/U). In conclusion, EliA anti-dsDNA assay showed a higher sensitivity than the CLIFT, and a good specificity and PPV for SLE. Its putative ability to detect only high-avidity anti-dsDNA antibodies remains questionable. J. Clin. Lab. Anal. 16:227,232, 2002. © 2002 Wiley-Liss, Inc. [source] Expression and characterization of the periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicidaJOURNAL OF FISH DISEASES, Issue 9 2009R Boiani Abstract Cobalamin (vitamin B12) is an essential cofactor in a variety of enzymatic reactions and most prokaryotes contain transport systems to import vitamin B12. A gene coding for a periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida was identified by in silico analysis of sequences from a genomic library. The open reading frame was composed of 834 bp encoding a protein of 277 amino acids. The protein showed 61% identity with the vitamin B12 -binding protein precursor of P. profundum, 53% identity with the corresponding protein of Vibrio parahaemolyticus and 43% identity with the periplasmic binding protein BtuF of Escherichia coli. The expression of the native protein was investigated in P. damselae subsp. piscicida, but BtuF was weakly expressed under normal conditions. To characterize the BtuF of P. damselae subsp. piscicida, the recombinant protein was expressed with a C-terminal His6 -tag and purified; the molecular weight was estimated to be approximately 30 kDa. The protein does not contain any free thiol group, consistent with the view that the two cysteine residues are involved in a disulphide bond. The purified BtuF binds cyanocobalamin with an affinity constant of 6 ± 2 ,m. [source] Biomimetic affinity purification of cardiotoxin and its pharmacological effects on the nervous system,JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2008Dexian Dong Abstract Cobra venom is a very precious natural resource. The traditional method for purification of cardiotoxin from cobra venom is a multi-step, high cost, and low recovery procedure. By molecular modeling and docking with SYBYL software, we designed and synthesized an affinity ligand, m-aminobenzoic acid, for high efficiency purification of this therapeutically useful Chinese cobra venom cardiotoxin. The one-step recovery of cardiotoxin reached 64% and the purity reached 92% upon purification. The binding capacity of this synthetic ligand was 9.1,mg cardiotoxin/g moist weight gel and the affinity constant for cardiotoxin was 5.5,×,103,M,1. Unlike a natural affinity ligand, this synthetic ligand is highly stable, and has great potential for industrial scale production of cardiotoxin. In addition, we examined the effects of cardiotoxin on the nervous system in a mouse model. Results showed that cardiotoxin could maintain analgesic effects for 120,min with a dose of less than 0.06,mg/kg (2.8% of the LD50). Administration of 0.12,mg/kg cardiotoxin could improve scopolamine impairments of memory in mice. These results suggest that cardiotoxin may be a potential drug for nervous system diseases. Copyright © 2008 John Wiley & Sons, Ltd. [source] Parameters of drug antagonism: re-examination of two modes of functional competitive drug antagonism on intraocular musclesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 8 2004Popat N. Patil There are two distinct kinetic functional pharmacological procedures by which the equilibrium affinity constant, KB, of a competitive reversible blocker is obtained. The classical method on an organ system requires the study of the parallel displacement of the agonist concentration-response curve in the presence of the blocker. In the second method, the agonist-evoked functional mechanical response is reduced to half by the blocker IC50 (the concentration required for 50% inhibition). In relation to these parameters the role of the ionization constant pKa and liposolubility log Pc or log D of blockers was examined. On the ciliary muscle from human eye, IC50/KB ratios for (±)-atropine, its quaternary analogue (±)-methylatropine, (-)-scopolamine, (±)-cyclopentolate, (-)-tropicamide, (±)-oxybutynin and pirenzepine were 15, 23, 4.4, 2.6, 1.66, 1.46 and 1.71, respectively. The ratios on the iris sphincter were comparable with those of ciliary muscle. When compared with large proportions of ionized molecules with water soluble properties of (±)-atropine and (±)-methylatropine, relatively high amounts of un-ionized and/or with greater partitioning of all other blockers in the lipoid barrier co-related well to low IC50/KB ratios, as predicted by the classical theory of competitive drug antagonism. It was hypothesized that due to receptor biophase access, the reduction of the mechanical response of the agonist by the highly ionized water-soluble antagonist at IC50 represented time-distorted "pseudo-equilibrium" estimation, where a higher concentration of the blocker was needed. On the other cholinergic effectors, like that of rat anococcygeus muscle or frog rectus abdominus muscle, IC50/KB ratios of respective blockers atropine or (+)-tubocurarine and hexamethonium were close to 1. Thus physicochemical properties, which affect the distribution coefficient log D and the tissue morphology (where asymmetric distribution of receptors may occur), appeared to be a critical factor in the analysis of the affinity parameters of the competitive reversible blocker. On the intraocular muscles, two functional pharmacological procedures for obtaining KB and IC50 values were not kinetically equivalent. [source] Identification of a ligand for IgG-Fc derived from a soluble peptide library based on fusion proteins secreted by S. cerevisiaeBIOTECHNOLOGY JOURNAL, Issue 6 2007Christa Mersich Abstract Biological libraries are important tools in the development of new peptide-based compounds. Here, we describe the use of a soluble peptide library system as a complementary tool in the field of ligand development. Random peptides were expressed in S. cerevisiae as carboxy-terminal extensions of the eukaryotic initiation factor 5a (eIF5a) and secreted into the culture supernatant. Expression and screening of this library were performed in a microwell format. As an example of this versatile approach, we describe the identification of a ligand for the human IgG-Fc fragment. Ligands binding IgG-Fc show great potential in a wide variety of applications including development of therapeutics, streamlining the large-scale purification of antibodies, and applications in diagnostic tests. We demonstrated the utility of this system. After screening only 6160 clones, we identified a ligand with the peptide sequence of TRRRTCSPPTWPRARARSTPSGCSSTGPSANRG. An affinity constant of 3.9 x 105 M -1 was determined by a biosensor method. Handling and maintenance of this library is conceptually simple and highly applicable for automated high-throughput systems. [source] Characterization of the in vitro adherence behavior of ultrasound responsive double-shelled microspheres targeted to cellular adhesion moleculesCONTRAST MEDIA & MOLECULAR IMAGING, Issue 6 2006Susanne Ottoboni Abstract We have developed novel adhesion molecule-targeted double-shelled microspheres which encapsulate nitrogen. We report in vitro targeting studies utilizing these microspheres conjugated to target-specific antibodies directed towards ICAM-1 and VCAM-1. In static adherence experiments, the adherence patterns of microspheres conjugated to three different monoclonal antibodies (two targeted to ICAM-1 and one to VCAM-1) to their target surfaces were very different. Maximum microsphere adherence at the lowest target and/or ligand densities was observed with the VCAM-1 system. Differences in target-specific adherence were also observed between anti-ICAM-1 and anti-VCAM-1 microsphere conjugates in flow adherence studies. Equilibrium binding studies of the target proteins in solution to the microsphere-bound ligands showed that the affinity constants of two microsphere-bound monoclonal antibodies for their target proteins are similar. Thus, ligand,target affinity is not the only determinant of microsphere adherence to the target surface in our systems. Shear stress was found to have an effect on the mean diameter of adhered microspheres; a decrease in the mean diameter with increasing shear was observed. The magnitude of this effect was dependent on both microsphere-bound ligand and target surface densities, with a more pronounced change at lower densities. Adhered microspheres were readily detectable using ultrasound at the lowest tested surface density of 40,mm,2. Copyright © 2006 John Wiley & Sons, Ltd. [source] Evaluation of enantioselective binding of antihistamines to human serum albumin by ACEELECTROPHORESIS, Issue 15 2007María Amparo Martínez-Gómez Abstract The drug binding to plasma and tissue proteins is a fundamental factor in determining the overall pharmacological activity of a drug. HSA, together with ,1 -acid glycoprotein, are the most important plasma proteins, which act as drug carriers, with implications on the pharmacokinetic of drugs. Among plasma proteins, HSA possesses the highest enantioselectivity. In this paper, a new methodology for the study of enantiodifferentiation of chiral drugs with HSA is developed and applied to evaluate the possible enantioselective binding of four antihistamines: brompheniramine, chlorpheniramine, hydroxyzine and orphenadrine to HSA. This study includes the determination of affinity constants of drug enantiomers to HSA and the evaluation of the binding sites of antihistamines on the HSA molecule. The developed methodology includes the ultrafiltration of samples containing HSA and racemic antihistaminic drugs and the analysis of the free or bound drug fraction using the affinity EKC-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of antihistamines to HSA, the major plasmatic protein. [source] Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresisELECTROPHORESIS, Issue 12 2004Mikael Nilsson Abstract A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using ,-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors. [source] Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galliFEBS JOURNAL, Issue 1 2005Rositsa Jordanova Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, ,-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N,-dimethyl- N -(iodoacetyl)- N,-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids. [source] Escherichia coli thioredoxin inhibition by cadmiumFEBS JOURNAL, Issue 7 2004Asp2, Two mutually exclusive binding sites involving Cys3 Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd2+ led us to investigate the thioredoxin,cadmium interaction properties. We used calorimetric and spectroscopic methods at different pH values to explore the relative contribution of putative binding residues (Cys32, Cys35, Trp28, Trp31 and Asp26) within or near the active site. At pH 8 or 7.5 two binding sites were identified by isothermal titration calorimetry with affinity constants of 10 × 106 m,1 and 1 × 106 m,1. For both sites, a proton was released upon Cd2+ binding. One mole of Cd2+ per mole of reduced thioredoxin was measured by mass spectrometry at these pH values, demonstrating that the two binding sites were partially occupied and mutually exclusive. Cd2+ binding at either site totally inhibited the thiol,disulfide transferase activity of Trx. The absence of Cd2+ interaction detected for oxidized or alkylated Trx and the inhibition of the enzymatic activity of thioredoxin by Cd2+ supported the role of Cys32 at the first site. The fluorescence profile of Cd2+ -bound thioredoxin differed, however, from that of oxidized thioredoxin, indicating that Cd2+ was not coordinated with Cys32 and Cys35. From FTIR spectroscopy, we inferred that the second site might involve Asp26, a buried residue that deprotonates at a rather high and unusual pKa for a carboxylate (7.5/9.2). The pKa of the two residues Cys32 and Asp26 have been shown to be interdependent [Chivers, T. P. (1997) Biochemistry36, 14985,14991]. A mechanism is proposed in which Cd2+ binding at the solvent-accessible thiolate group of Cys32 induces a decrease of the pKa of Asp26 and its deprotonation. Conversely, interaction between the carboxylate group of Asp26 and Cd2+ at a second binding site induces Cys32 deprotonation and thioredoxin inhibition, so that Cd2+ inhibits thioredoxin activity not only by binding at the Cys32 but also by interacting with Asp26. [source] Direct monitoring of molecular recognition processes using fluorescence enhancement at colloid-coated microplatesJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2001Ch. Lobmaier Abstract Direct monitoring of recognition processes at the molecular level is a valuable tool for studying reaction kinetics to assess affinity constants (e.g. drugs to receptors) and for designing rapid single step immunoassays. Methods currently used to gain information about binding processes predominantly depend on surface plasmon resonance. These systems use excitation with coherent light in attenuated total reflection geometry to obtain discrimination between surface-bound and free molecules in solution. Therefore labeling of the compounds is not necessary, but due to the complexity of the measuring setup the method is rather costly. In this contribution we present a simple method for performing kinetic single step biorecognition assays with fluorophore labeled compounds using the fluorescence enhancement properties of surface bound silver colloids. Silver colloids are bound to standard microplates via silanization of the plastic surface. Fluorophores close to this colloid coated surface show a significant gain in fluorescence compared to fluorophores farther away in the bulk solution. Therefore discrimination between surface bound and free fluorophores is possible and the binding of, for example, fluorophore labeled antibodies to antigens immobilized on the colloid surface results in increasing fluorescence intensity. Utilization of standard microplates makes this method fully compatible with conventional microplate processing and reading devices. Neither excitation with coherent laser light nor ATR geometry is required, the measurement is performed in a standard fluorescence microplate reader in front face geometry with a xenon flash lamp as excitation source. Methods for the preparation of colloid-coated microplates and fluorescence-enhanced biorecognition assays are presented. Additionally the dependence of the system performance on the structure and properties of the metal colloid coated surface is described. A two-component biorecognition model system shows a detection limit in the subnanomolar range. The ease of colloid-surface preparation and the high sensitivity makes fluorescence enhancement at colloid-coated microplates a valuable tool for studying reaction kinetics and performing rapid single-step immunoassays. Copyright © 2001 John Wiley & Sons, Ltd. [source] Responsiveness, affinity constants and receptor reserves for serotonin on aortae of aged normotensive and hypertensive ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2001Sheila A. Doggrell We have previously shown that the potency and affinity constants (KA values) for serotonin (5-HT) are greater, and the 5-HT2A -receptor reserve is lesser, on the aorta of 6-month-old spontaneously hypertensive rats (SHRs) compared with age-matched Wistar Kyoto normotensive (WKY) rats. The present study was undertaken to investigate whether these parameters are altered on the aorta with ageing and as hypertension progresses to heart failure. The effects of phenoxybenzamine on the serotonergic responses of the aortae of 24-month-old WKY rats and SHRs were determined. On WKY rat aorta, ageing from 6 to 24 months was associated with an increase in sensitivity and affinity for serotonin, and a loss of 5-HT2A -receptor reserve. On SHR aorta, ageing from 6 to 24 months was also associated with an increase in sensitivity and affinity for serotonin, but a loss of 5-HT2A -receptor reserve. The sensitivity to serotonin was greater on the 24-month-old SHR aorta (pD2 6.53) than age-matched WKY rat aorta (pD2 5.89). On the aorta of the 24-month-old WKY rats, the KA value for serotonin was 4.5 times 10,6 M, and the receptor occupancies required for 20 and 50% maximum responses were 12 and 29%, respectively. There was a similar affinity, but greater receptor reserves, for serotonin on the aorta of age-matched SHRs. In summary, we have shown changes in sensitivity, affinity and 5-HT2A -receptor reserves for serotonin on the aorta with ageing and in hypertension/heart failure. [source] Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivatization reagentBIOMEDICAL CHROMATOGRAPHY, Issue 5 2006Yasuhiko Higashi Abstract Simultaneous HPLC assay of 1-adamantanamine hydrochloride (amantadine) and its four related compounds [2-adamantanamine hydrochloride (2-ADA), 1-adamantanmethylamine (ADAMA), 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) and 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] in phosphate-buffered saline (pH 7.4) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed. Phosphate-buffered saline samples were mixed with borate buffer and NBD-F solution in acetonitrile at 60°C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2-ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 µg/mL, respectively. The coefficients of variation for intra- and inter-day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human ,1 -acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD-F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre-column derivatization. Our method can be applied to binding studies for protein. Copyright © 2005 John Wiley & Sons, Ltd. [source] | ||||||||||||||||