Regular ArticleThe Problem of Partial Competition in the Quantitative Characterization of Interactions by Competitive Binding Assays
Abstract
Binding expressions are derived and analytical procedures developed for the quantitative characterization of inhibitor binding that is only partially competitive with the interaction between an acceptor and the ligand that is being monitored. Two such situations are considered: (i) that in which the partial competition reflects binding of inhibitor to fewer acceptor sites than available to ligand; and (ii) that in which the partial competition reflects binding of inhibitor to acceptor sites in addition to those occupiable by ligand. The potential efficacy of the suggested analyses is then explored by their application to simulated data that span the likely range of experimental behavior. Quantitative analysis of the displacement of [3H]nitrobenzylthioinosine from cultured leukemic cells by an adduct of 5′-S-(2-amino-ethyl)-N6-(4-nitrobenzyl)-5′-thioadeflosine with fluorescein-5-isothiocyanate is used to establish that the cells posses 6% fewer sites (150,000 cf. 159,000 sites/cell) for the fluorescent adduct than for the tritiated ligand. and that the binding is 10-fold weaker (binding constant of 0.28 cf. 2.8 nM−1). Corresponding analysis of results obtained with bovine aorta endothelial cells indicates that a 3-fold weaker interaction (binding constant of 1.1 cf. 3.3 nM−1) occurs between the fluorescent adduct and 79% of the cell sites accessible to the tritiated ligand. The present analytical procedures extend the utility of competitive binding assays for the quantitative screening of potential inhibitors by removing the inherent limitation of existing analyses that all acceptor sites be accessible to both the competing solute and the indicator ligand.
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Determination of binding constants by affinity chromatography
2004, Journal of Chromatography AThis review summarizes developments in the use of affinity chromatography to characterize biospecific interactions in terms of reaction stoichiometry and equilibrium constant. In that regard, the biospecificity incorporated into the design of the experiment ensures applicability of the method regardless of the sizes of the reacting solutes. By the adoption of different experimental strategies (column chromatography, simple partition equilibrium, solid-phase immunoassay and biosensor technology protocols) quantitatiative affinity chromatography can be used to characterize interactions governed by an extremely broad range of binding affinities. In addition, the link between ligand-binding studies and quantitative affinity chromatography is illustrated by means of partition equilibrium studies of glycolytic enzyme interactions with muscle myofibrils, an exercise which emphasizes that the same theoretical expressions apply to naturally occurring examples of affinity chromatography in the cellular environment.
Synthesis and evaluation of affinity adsorbents for glycoproteins: An artificial lectin
2000, Journal of Chromatography B: Biomedical Sciences and ApplicationsA combination of rational design based on mimicking natural protein–carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, 1H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 μmol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M α-d-methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for α-d-mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of α-l-fucose. Interestingly, α-l-fucose and α-d-mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of d-mannose, d-glucose, l-fucose and d-galactose with immobilised 18/18 establish that the affinity constants (KAX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3×105 M−1, 1.9×104 M−1 and 1.2×104 M−1 respectively. 1H-NMR studies on the interaction of α-d-methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of α-d-methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.
Flow cytometric studies of nucleoside transport regulation in single chromaffin cells
1998, FEBS LettersThe present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [3H]NBTI binding with a Ki of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (KD=6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.
The rectangular hyperbolic binding equation for multivalent ligands
1995, Archives of Biochemistry and BiophysicsAn improved procedure for the characterization of interactions involving multivalent ligands which eliminates a previous requirement that interaction parameters for independent and equivalent binding be evaluated from a general counterpart of the Scatchard analysis is described. Binding data for the interaction of aldolase with muscle myofibrils are then used to illustrate application of the procedure, which allows the stoichiometry and intrinsic binding constant to be evaluated by standard nonlinear regression analysis in terms of a rectangular hyperbolic relationship.
General treatment of competitive binding as applied to the potentiometric ion probe technique: Application to the interaction of nonsteroidal anti‐inflammatory drugs with bovine serum albumin
1994, Journal of Pharmaceutical SciencesThe binding of naproxen, ketoprofen, phenylbutazone, salicyclic acid, azapropazone, and indobufen to bovine serum albumin was studied by applying the potentiometric ion probe technique. An ion-selective electrode for the ion probe 1-anilino-8-naphthalene-sulfonate was utilized for the purposes of this study. A modified site-oriented competitive binding model was used for the estimation of the drugs' binding parameters, considering different number of binding sites on the competing binding class(es) for the probe and the drug. Calculations were based exclusively on the concentration data of the free probe. The model's ability for accurate estimations of binding parameters was evaluated by simulation studies. The following values of binding parameters were found at 25 °C for the drugs under study; naproxen, n1 = 9.1, k1 = 9.4 × 105 M− 1; ketoprofen, n1 = 8.8, k1 = 10.8 × 105 M− 1; phenylbutazone, n1 = 3.2, k1 = 1.4 × 105 M− 1; salicylic acid, n1 = 2.6, k1 = 1.8 × 105 M− 1, n2 = 21.5, k2 = 1.0 × 104 M− 1; azapropazone,n1 = 0.5, k1 = 7.8 × 105, M− 1, n2 = 26.3, k2 = 1.9 × 104 M− 1; indobufen, n1 = 5.8, k1 = 5.8 × 105 M− 1, n2 = 19.9, k2 = 3.8 × 105 M− 1, where ni the number of binding sites of the i class and ki, the corresponding association constant.