Binding Studies on Resins Imprinted with (S)-naproxen

2002 ◽  
Vol 723 ◽  
Author(s):  
Yue Hu ◽  
Robert A. Orwoll
Keyword(s):  
Free Radical ◽  
Binding Sites ◽  
Affinity Binding ◽  
Binding Parameters ◽  
Glycol Dimethacrylate ◽  
Binding Studies ◽  
Number Of Binding Sites ◽  
Scatchard Equation ◽  
Non Covalent Interactions ◽  
Covalent Interactions

AbstractResins were prepared in a free-radical polymerization of 4-vinylpyridine and ethylene glycol dimethacrylate in the presence of (S)-(+)-6-methoxy-α-methyl-2-naphtaleneacetic acid ((S)-naproxen). Initially (S)-naproxen, the imprinted molecule template, was assembled with the monomer 4-vinylpyridine by non-covalent interactions. After the polymerization, stepwise removal of the template left binding sites that retain complementary specificity and affinity. Binding parameters including the maximum number of binding sites and dissociation constant were calculated from the amount of template removed using a two-site Scatchard equation. The results are typical of other systems reported in the literature.

Adrenergic receptors on cerebral microvessels: pericyte contribution

1989 ◽  
Vol 256 (1) ◽  
pp. R224-R230 ◽  
Author(s):  
R. M. Elfont ◽  
P. R. Sundaresan ◽  
C. D. Sladek
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Studies ◽  
Cerebral Microvessels ◽  
Beta Adrenoceptor ◽  
Number Of Binding Sites

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

Interfaces of Molecular Recognition Sites and Biosensors; Novel Bio-Sensing Materials

2015 ◽  
Vol 1793 ◽  
pp. 1-6
Author(s):  
Kyung M. Choi
Keyword(s):  
Binding Sites ◽  
Glass Surface ◽  
High Sensitivity ◽  
Affinity Binding ◽  
Sensing Element ◽  
Practical Applications ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Number Of Binding Sites ◽  
Low Sensitivity

ABSTRACTWe introduce a sensing element, “Molecularly Imprinted Polymer (MIP),” which created by “Molecular Imprinted Technique.” However, the sensitivity of MIP’s based bio-sensors limits for practical applications due to the low sensitivity. To achieve a high sensitivity of MIP’s based sensors, the synthesis of “high affinity receptor or binding sites,” such as “monoclonal particles” is a key objective. In previous studies, affinity distribution plots indicated that “high affinity binding sites” were obtained when the number of binding sites per particle decreased. It means that smaller particles are expected to have higher affinity binding sites compared to larger particles. The result motivated us to produce small-sized MIP’s particles for the achievement of higher sensitivity. Microfluidic Synthesis has taken a great attention to synthesize small particles. However, the microfluidic synthesis gave us a difficulty, especially collections of MIP’s particles from the surface of PDMS-based microchannels due to a sticking problem. Thus, we employed a new approach, which can collect MIP’s particles without any sticking problem from the surface of the reactor. It is a photopatterned MIP’s system generated on the glass surface. We prepared a photomask with micro-sized patterns and then fabricate MIP’s particles on a glass surface by photopolymerization. Uniform MIP’s patterns were printed on the glass surface. The interface between the glass surface and the MIP’s pattern was observed by SEM. Micro-sized MIP’s particles were collected from the glass surface by scratching off the photocured MIP’s patterns.

scholarly journals Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms

1986 ◽  
Vol 240 (2) ◽  
pp. 403-412 ◽  
Author(s):  
E Kloprogge ◽  
J W Akkerman
Keyword(s):  
Platelet Activation ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
A Minor

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [(3H]arachidonate mobilization and thromboxane synthesis) and secretion [(14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.

The ‘ligand-induced conformational change’ of alpha 5 beta 1 integrin. Relocation of alpha 5 subunit to uncover the beta 1 stalk region

1998 ◽  
Vol 111 (12) ◽  
pp. 1759-1766 ◽  
Author(s):  
J. Tsuchida ◽  
S. Ueki ◽  
Y. Takada ◽  
Y. Saito ◽  
J. Takagi
Keyword(s):  
Conformational Change ◽  
Binding Sites ◽  
K562 Cells ◽  
Affinity Binding ◽  
Fab Fragment ◽  
High Affinity Binding ◽  
High Affinity ◽  
Number Of Binding Sites ◽  
Beta 1 Integrin ◽  
Beta1 Subunits

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.

Molecular Imprinting of 3-Hydroxybenzoic Acid: Special and General Binding Sites

2003 ◽  
Vol 787 ◽  
Author(s):  
Yue Hu ◽  
Robert A. Orwoll
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Crosslinking Agent ◽  
The Other ◽  
Hydroxybenzoic Acid ◽  
Functional Monomer ◽  
Glycol Dimethacrylate ◽  
Imprinted Polymer ◽  
Site Model ◽  
Scatchard Equation

ABSTRACTA resin, imprinted with 3-hydroxybenzoic acid (3HBA), was synthesized from acrylamide (AA, the functional monomer) and ethylene glycol dimethacrylate (EGDMA, the crosslinking agent). Batch analyses showed that the imprinted polymer has a special affinity for the meta-substituted 3HBA, but not for its para-substituted isomer (4HBA) nor for benzoic acid (BA). These results are consistent with the principle that an imprinted resin's ability to recognize is dependent on the target's size, shape, and functionality. Another resin, prepared from AA and EGDMA but in the absence of a template, had similar affinities for 3HBA, 4HBA, and BA; and thus it could not differentiate among the three. The results can be interpreted with a simple two-binding-site model with one site special for 3HBA and the other being more general with similar affinities for 3HBA, 4HBA and BA. The binding of 3HBA to the imprinted resin is characterized by an association constant and the density of each kind of site using a two-site Scatchard equation. The binding sites common to both the imprinted resin and the non-imprinted reference resin were found to have greater affinity but are less numerous than the sites unique to the imprinted resin.

A New Method To Determine The Binding Of Factor X To Phospholipid Bilayers

1981 ◽  
Author(s):  
G Tans ◽  
J Rosing ◽  
G V Dieijen ◽  
J V Rijn ◽  
H C Hemker
Keyword(s):  
Binding Sites ◽  
Phospholipid Bilayers ◽  
Phospholipid Bilayer ◽  
Activate Factor ◽  
Factor X ◽  
Binding Parameters ◽  
Clotting Factors ◽  
Number Of Binding Sites ◽  
Ca Ions ◽  
Factor X Activation

The binding parameters (dissociation constant and the number of binding sites) for factor X binding to phospholipid bilayers in the presence of Ca-ions can be determined using the factor X-activating protein present in Russel’s Viper Venom (RVV-X). It is shown that RVV-X is only able to activate factor X molecules that are not bound to the phospholipid bilayer. Therefore, from the observed rates of factor X activation by RVV-X measured in the presence and absence of phospholipid the amount of factor X not bound to the phospholipid bilayer can be calculated. When the concentration of factor X added is known the bound factor X can be calculated and the binding parameters for factor X are obtained from a Scatchard plot. The technique presented here offers an advantage over other techniques published thus far. It is rapid and simple as compared to the Hummel andDryer technique. There is also an advantage over techniques using light scattering. Since the technique presented here is not hampered by aggregation of vesicles it is possible to study the binding of factor X to vesicles containing a high mole fraction of PS and at high CaCl2 concentrations.The binding parameters for factor X binding to vesicles of a mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) are dependent on the amount of negatively charged phospholipid present. At increasing mole fractions of PS factor X binds more tightly to the vesicles and also the number of factor X binding sites increases. However, at high amounts of PS (60%) the number of sites present decreases dramatically which is likely due to aggregation of the vesicles. Binding to vesicles composed of a mixture of phosphatidylgly- cerol (PG) and PC is considerably weaker whereas the lumber of sites present is about the same. Prothrombin and factor X compete for the same binding site as was determined by competition experiments. Our data will be discussed with respect to the mode of interaction of vitamin-K dependent clotting factors with negatively charged phospholipid interfaces.

High-affinity calcium binding sites in luminal and basolateral renal membranes

1985 ◽  
Vol 248 (4) ◽  
pp. F472-F481
Author(s):  
Z. Talor ◽  
G. Richison ◽  
J. A. Arruda
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Divalent Cations ◽  
Ruthenium Red ◽  
Affinity Constant ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Ca Uptake ◽  
Number Of Binding Sites

We evaluated Ca binding by highly purified luminal (L) and basolateral (BL) tubular membranes prepared from beef kidney. Ca binding was measured by using 45Ca and a rapid-filtration technique. After Ca uptake reached equilibrium, the vesicles were lysed and the amount of 45Ca retained in the membranes was considered the bound Ca. Ca binding in both membranes accounted for approx. 80% of total Ca uptake. Analysis of binding data by Scatchard plot revealed the presence of two distinct types of binding sites in both L and BL membranes. The high-affinity binding sites showed a similar affinity constant of 10(-5)M for both L and BL membranes, but the maximum number of binding sites was 0.75 and 1.6 nmol/mg protein, respectively. In contrast, the low-affinity binding sites were similar regarding affinity constant and maximum number of binding sites in the two membranes. In L and BL membranes, high-affinity binding sites were selective for Ca, as high concentrations of divalent cations were required to inhibit Ca binding. In both membranes Ca binding was inhibited by ruthenium red, LaCl3, and detergents, and it was stimulated by calmodulin inhibitors (trifluoperazine, calmidazolium), ionophore A-23187, and ATP. These results demonstrate that L and BL membranes possess high-affinity binding sites with different capacities but similar characteristics as regards affinity constant and stimulation and inhibition of binding. The data further demonstrate that most of Ca uptake by these membranes represents binding.

scholarly journals Determination of the Binding Parameters between Proteins and Luminol by Chemiluminescence Using Flow Injection Technique

2013 ◽  
Vol 2013 ◽  
pp. 1-5
Author(s):  
Jie Guo ◽  
Donghua Chen ◽  
Zhenghua Song
Keyword(s):  
Bovine Serum Albumin ◽  
Flow Injection ◽  
Binding Sites ◽  
Bovine Serum ◽  
Binding Constants ◽  
Injection Technique ◽  
Binding Parameters ◽  
Hydrophobic Force ◽  
Number Of Binding Sites

The interaction behavior of bovine serum albumin (BSA), lysozyme (LYS), myoglobin (MB), and catalase (CAT) with luminol, respectively, was first studied by chemiluminescence (CL) using flow injection (FI) technique based on the fact that the studied proteins can enhance the CL intensity of luminol. A FI-CL model of protein-luminol interaction, lg[(I0−I)/I]=1/nlg[P]+1/nlgKa+2lgn, was constructed, and the interaction parameters of BSA, LYS, MB, and CAT with luminol were determined accordingly. The binding constants Ka are in the descending order of CAT > MB > LYS > BSA at the level of 105 to 107 L mol−1, and the number of binding sites n of luminol to BSA or LYS is around 2 and to MB or CAT is around 1. The results of thermodynamic parameters (ΔH, ΔS, and ΔG) showed that the binding processes of luminol to the four proteins are spontaneous mainly through the hydrophobic force.

scholarly journals Biophysical cytochemical investigations of intracellular heparin in neoplastic mast cells.

1980 ◽  
Vol 28 (3) ◽  
pp. 223-230 ◽  
Author(s):  
N Nakamura ◽  
R E Hurst ◽  
S S West ◽  
J M Menter ◽  
J F Golden ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Binding Constant ◽  
Binding Constants ◽  
Cooperative Binding ◽  
Binding Parameters ◽  
Binding Studies ◽  
Mastocytoma Cells ◽  
Biochemical Analyses

The thermodynamic binding parameters for intracellular heparin-acridine orange (AO) complexes were determined for Furth murine mastocytoma cells and were found to agree with 1) results from binding studies on heparin-AO complexes in solution, and 2) with biochemical analyses of the cells. The cells exhibited cooperative binding with a binding constant of 1.18 x 10(6) M-1. The cooperative binding constant of heparin-AO in 1 mM buffer was found to be 1.13 x 10(6) M-1. The addition of 1 mM NaCl to heparin-AO system in vitro detectably decreased the cooperative binding constant. Low ionic strength is the only condition in solution under which the cell and solution binding constants are equal. The cells have an average of 1.2 x 10(-14) mol of AO binding sites per cell. Using the biochemically measured heparin content per cell and the amount of AO bound by heparin in solution, 8 x 10(15) mol of sites/cell can be attributed to heparin. The remaining cellular binding sites (4 x 10(-15) mol of sites per cell) are essentially all accounted for by AO binding to DNA, the amount of which is calculated from its previously determined thermodynamic binding parameters. A theoretical isotherm, calculated from the binding parameters of both heparin-AO in solution and DNA-AO complexes in situ, agreed closely with the isotherm experimentally determined for the Furth mastocytoma cells. Ligand-binding analysis yields a binding constant, which may aid in identification of cellular bipolymers, and the number of ligand binding sites per cell. The latter is a measure of the amount of a given intracellular biopolymer present.

Postnatal changes in the substance P receptor on rabbit gastric smooth muscle

1992 ◽  
Vol 262 (2) ◽  
pp. G291-G297
Author(s):  
P. E. Hyman ◽  
S. Kimura ◽  
T. Tomomasa ◽  
Q. X. Yuan ◽  
W. J. Snape ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Substance P ◽  
Binding Sites ◽  
Tissue Homogenate ◽  
Neurokinin A ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gastric Smooth Muscle

We used radioligand binding to tissue homogenates and isometric contraction of muscle strips to characterize the substance P (SP) receptor on gastric smooth muscle from 1- (newborn) and 7-day-old and 4- and 11-wk-old (weanling) rabbits. Scatchard analysis for newborns was curvilinear, suggesting the presence of multiple binding sites. In newborns the dissociation constant (Kd) of high-affinity binding site was 2.2 +/- 0.3 nM, and the maximum binding (Bmax) was 0.57 +/- 0.06 pmol/mg DNA. The number of high-affinity binding sites decreased with age, disappearing by 11 wk. The Kd for the low-affinity site was more than two orders of magnitude greater than that of the high-affinity site. In competitive binding studies with [3H]SP, the order of potency for the neurokinins was SP much greater than neurokinin A (NKA) greater than neurokinin B (NKB), suggesting that the high-affinity binding sites were NK-1 receptors. [125I]NKA is also bound to newborn tissue homogenate with high affinity. With [125I]NKA the order was NKA greater than SP greater than NKB, suggesting that NK-2 receptors were also present. In contraction studies, atropine and tetrodotoxin had no effect on tachykinin-stimulated contraction, suggesting solely myogenic tachykinin effects on this tissue. In newborn rabbits, the potency and efficacy of SP and NKA were similar. The half-maximal effective dose (ED50) of SP was nearly two orders of magnitude less in newborn rabbits than in weanlings; the potency of NKA did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

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